Inhibitors of the notch signalling pathway and secretion for use in medicine

ABSTRACT

The invention relates to dihydropyridine compounds as inhibitors of the notch signalling pathway and/or inhibitors of secretion for the treatment of secretion-dependent disease, such as cancer or senescence-related ageing, in addition to pharmaceutical compositions thereof and methods of treatment.

The invention relates to chemical compounds as inhibitors of the notchsignalling pathway and/or secretion for the treatment ofsecretion-dependent disease, such as cancer or senescence-relatedageing, in addition to pharmaceutical compositions thereof and methodsof treatment.

BACKGROUND OF THE INVENTION

Notch is an important regulator of gene transcription involved incellular differentiation, lateral inhibition and tissue homeostasis. InNotch signaling the Notch precursor protein is activated in thetrans-Golgi-network by proteolytic cleavage. This cleavage generates twofragments that comprise the active Notch-receptor, which is thentranslocated to the plasma membrane (PM). Upon interaction with itsligand from the Delta/Serrate/Jagged family it is subsequently processedby ADAM 17 and finally by gamma-secretase to generate the Notchintracellular domain (NICD). NICD is translocated to the nucleus, whereit activates a transcription factor of the CSL family (CBF1 (RBP-J) invertebrates, Su(H) in Drosophila, Lag-1 in C. elegans). NICD is composedof several Ankyrin repeats and an N-terminal RAM domain, which iscritical for the activation of CSL (for review see¹). Furthermore, onecharacteristic and important hallmark of the Notch signaling pathway isthe independence of second messengers. Thus, the amount of NICD insidethe nucleus is proportional to the amount of processed Notch receptor.

The final cleavage step that releases NICD from the PM is mediated bygamma-secretase. gamma-secretase is a heteromultimeric integral membraneprotein with protease activity². Interacting and potentially regulatingproteins were shown³⁻⁶ but a commonly accepted view of the regulation ofgamma-secretase is lacking. Notch signaling can be fine-tuned at severalsteps, ranging from glycosylation at the ectodomain, interaction withinhibitors like Numb, ubiquitinylation and phosphorylation of NICD toslight variations at the gamma-secretase cleavage site (for reviewsee¹). Fine-tuning can regulate trafficking of the receptor, ligandbinding-affinity, endocytosis rate of the receptor and stability ofNICD^(1,7). Despite the wealth of information about Notch signaling, itstrafficking and regulation of signaling is not fully understood.

Therapeutic interventions of aberrant Notch signaling, for example incases of T cell-lineage acute lymphoblastic leukemia (T-ALL) at presentare aimed/tested at the level of ligand-binding and gamma-secretasecleavage, but novel strategies would be highly desired^(8,9).

Natural compounds like Brefeldin A (BFA), Shiga- or Cholera-toxin turnedout to be invaluable tools to dissect molecular details of membranetrafficking at various steps in the exo- or endocytic pathway¹⁰.

Similarly, gamma-secretase related research could not have come this farwithout the identification of gamma-secretase inhibitors (GSI) such asDAPT or L685,458. These and other GSIs turned out to be not onlyinvaluable to inhibit gamma-secretase but also to purify it, elucidatestructure-function relations within the gamma-secretase complex andidentify substrate-docking sites (13).

High content screening (HCS) describes the process of automated imageacquisition of phenotypes of cells or organisms and the subsequentautomated analysis of these phenotypes by image analysis algorithmswithout or only little user intervention allowing for high throughputapplications such as genome-wide RNAi or large chemical compoundlibraries (for review see 14).

Despite various innovative screening strategies having been applied,there is a need in the field to find further inhibitors of the notchsignaling pathway for application in medicine.

GSIs have been applied in the treatment of cancer, based on the findingthat Notch signaling is misregulated in various cancers. The Notchsignalling pathway is an important component in the molecular mechanismsthat regulate cell fate during development, in addition to cancerformation. Aberrant activation of the Notch pathway contributes totumour formation. The important role of Notch in human cancer has beenhighlighted by the presence of activating mutations and amplification ofNotch genes in human cancer and by the demonstration that genes in theNotch signalling pathway could be potential therapeutic targets. One ofthe major therapeutic targets in the Notch pathway are the Notchreceptors, in which γ-secretase inhibitors prevent the generation of theoncogenic (intracellular) domain of Notch molecules and suppress Notchactivity. Further notch inhibitors are therefore sought after aspotential cancer therapeutics.

Despite the promising effects of gamma-secretase inhibitors, knowninhibitors are plagued by significant side effects during and aftertreatment of patients. Major side effects relate to gastrointestinaltoxicity, such as nausea, diarrhea, vomiting, weight loss and/or loss ofappetite.

The present invention therefore provides novel compounds not previouslyidentified as Notch inhibitors. Furthermore, the compounds of thepresent invention show an anti-secretion function and play a role inmembrane trafficking, inhibiting secretion at a pre-ER exit step. Theeffect of administration of the compounds of the invention inpre-clinical assays shows a notch phenotype in addition to disruption ofsecretion at an early stage. This effect enables the compounds asdisclosed herein to be administered in the treatment of diseasesassociated with secretion or secretion pathways.

One example of such a condition is senescence-related ageing, wherebysenescent cells lose their proliferative capacity once senescence occursand show an enhanced secretion of pro-inflammatory cytokines, which isalso known as the “senescence associated secretory phenotype (SASP):

Accumulation of nuclear DNA damage represents one of the molecularcauses of aging. Accordingly, there is an age-dependent accumulation ofDNA damage in numerous human tissues and genetic diseases that lead topremature aging are often caused by mutations in genes involved in DNAdamage repair; and animal models provided a proof of concept that DNAdamage accumulation leads to premature aging. Cells respond to DNAdamage by activating checkpoints that prevent the contribution ofdamaged cells to tissue homeostasis by induction of cell death(apoptosis), cell cycle arrest (senescence) or self-digestion of thedamaged cells (autophagy).

Senescent cells lose their proliferative capacity and show an enhancedsecretion of pro-inflammatory cytokines, which is also known as the“senescence associated secretory phenotype (SASP)”. It has been shownthat SASP affects neighbouring, non-senescent cells and this may impacton tissue aging and cancer formation. Methods or means for theinhibition of SASP could delay tissue dysfunction and extend healthylifespan.

Little is known about the SASP-underlying changes in structure andcomposition of the secretory pathway. Senescence is associated withmorphological changes in the secretory pathway like dispersal of theGolgi, increase in lysosomal volume, increase in lysosomal lipofuscinaggregation and increased expression of the lysosomal enzymeβ-galactosidase. In light of the secretion phenotype associated withSASP, inhibitors of secretion represent a promising means for treatingillness associated with SASP. At the present time no effectivetherapeutic approaches for the treatment of SASP-associated disease areknown, and appropriate secretion inhibitors for the treatment of suchdisorders are required.

The compounds of the present invention therefore relate to FLI-06 andstructurally similar derivatives that exhibit the properties desired incompounds for treatment of secretion-dependent disease, such as canceror ageing.

Structurally similar compounds have been previously disclosed inrelation to different biological effects or for the treatment ofdifferent medical conditions. WO 2009/102864 discloses structurallysimilar compounds to those of the present invention as antagonists ofthe hedgehog signaling pathway. However, the compounds disclosed thereinare not mentioned in relation to secretion or notch signaling inhibitionand additionally show different structural features, for example theester position of R2 of formula I of the present invention is distinctfrom those compounds of WO 2009/102864, which exhibit an alkoxymethoxyethyl group at the corresponding position.

WO 2008/103470 discloses structurally similar compounds to those of thepresent invention as RAS-specific anti-cancer agents. However, thecompounds disclosed therein are not mentioned in relation to secretionor notch signaling inhibition and additionally show different structuralfeatures, for example the ester position of R2 of formula I of thepresent invention is distinct from those compounds of WO 2008/103470,which exhibit an alkoxy group at the corresponding position.Furthermore, the compounds of WO 2008/103470 exhibit a phenylsubstituent in place of R6. Additionally the compounds disclosed thereinexhibit a different central ring structure compared to the compounds offormula I of the present invention.

WO 2011/050353 discloses structurally similar compounds to those of thepresent invention for the treatment of androgen-receptor-positive cancercells. However, the compounds disclosed therein are not mentioned inrelation to secretion or notch signaling inhibition and additionallyshow different structural features, for example the ester position of R2of formula I of the present invention is distinct from those compoundsof WO 2011/050353, which exhibit a phenylethyl group. It is noteworthythat none of the compounds disclosed in the art that exhibit structuralsimilarity to the presently claimed compounds and are intended forcancer treatment exhibit a substitution of the hexahydroquinoline ringat position 7 with two methyl groups.

WO 2008/070875 also discloses structurally similar compounds to those ofthe present invention. The compounds of WO 2008/070875 are onlydisclosed in relation to the treatment of Alzheimer's disease and arenot mentioned in relation to secretion or notch signaling inhibition.

SUMMARY OF THE INVENTION

The compounds of the invention were products of an assay established tomonitor membrane trafficking and processing of a ligand-independentNotch-GFP reporter by automated microscopy. 16.671 small compounds werescreened and relevant candidates validated by cellular and in vitroassays as well as by using zebrafish as an in vivo model. FLI-06functions by disrupting the Golgi apparatus, different from Brefeldin Aand Golgicide A. Detailed analysis reveals that FLI-06 inhibitssecretion at a pre-ER exit step, making it the first identified compoundthat blocks secretory traffic at such an early stage. The furtherderivatives of the invention as tested herein also demonstrate similardisruption of secretion and membrane trafficking. Molecularcharacterization of selected compounds identified novel inhibitors ofER-export, which have the unique property to inhibit cargo recruitmentto ER exit sites.

The invention is therefore based on a novel type of secretion inhibitor,FLI-06 and derivatives thereof. In contrast to other secretioninhibitors, no gross ER stress is induced. FLI-06 and its derivativesrepresent useful medicaments for inhibiting or reducing secretionwithout inducing ER-stress mediated apoptosis.

The combination of disruption of membrane traffic and secretion andnotch signalling inhibition, together with the treatment ofsecretion-dependent disease represents a novel and non-obviousdevelopment in the field. This combination of compound, mechanism andmedical use has not been suggested previously with regard to any of thepreviously known notch inhibitors. This combination (which is common tothe compounds of the present invention) represents a unique contributionto the prior art.

In light of the prior art the technical problem underlying the inventionwas the provision of compounds for the inhibition of notch signaling viainhibition of secretion for use in medicine, in particular providingpharmaceutically active compounds that exhibit fewer side effects thanknown notch inhibitors.

This problem is solved by the features of the independent claims.Preferred embodiments of the present invention are provided by thedependent claims.

The invention therefore relates to compounds according to the generalformula I as inhibitors of the notch signalling pathway for use as amedicament for the treatment of a secretion-dependent disease

whereby R1 is one of:

whereby X is H or a halogen, preferably F, Cl, Br or I, and Y isCOOCH₃or CF3, or COOH;R2 is a straight chain or branched alkyl group of C₁-C₈, or a carbonring structure of C₅-C₈, preferably one of

R3 is H or a straight chain or branched alkyl group of C₁-C₆;R4 is H or a straight chain or branched alkyl group of C₁-C₆, preferablya branched alkyl group selected from

R5 is H or a straight chain or branched alkyl group of C₁-C₆;whereby R4 and R5 may be closed with a C₃ group to form a C₆ ringstructure with the group

whereby R6 and/or R7 is H or a straight chain or branched alkyl group ofC₁-C₆;whereby “R” of the substituents R1, R2, R4, R5 represents the backboneof general formula I.

In a preferred embodiment of the invention the compounds of formula Iare further characterised by the general formula II

whereby R1-R3 are as defined by formula I, and R6 and/or R7 is H or astraight chain or branched alkyl group of C₁-C₆.

The invention therefore relates to formula I and/or II

whereby R1 is one of:

whereby X: H or a halogen, preferably F, Cl, Br or I, and Y: COOCH₃ orCF3, COOH,R2 is a straight chain or branched alkyl group of C₁-C₈, or a carbonring structure of C₅-C₈, preferably one of

R3 is H or a straight chain or branched alkyl group of C₁-C₆,R4 is H or a straight chain or branched alkyl group of C₁-C₆, preferablya branched alkyl group selected from

R5 is H or a straight chain or branched alkyl group of C_(r)C₆, andR6, R7 is H or a straight chain or branched alkyl group of C₁-C₆,whereby “R” of the substituents R1, R2, R4 represents the backbone ofgeneral formula I-a or I-b

In a preferred embodiment the compounds of the present invention arecharacterised by the general formula III

whereby R1: H, COOCH₃, COOH, CN, NO₂, CF₃, or NH₂ and R2: a straightchain or branched alkyl group of C₁-C₈, preferably C₂-C₆, or a carbonring structure, preferably C₅, C₆, C₇ or C₈.

The present invention relates additionally to compounds with a structureaccording to any one of formulae I, nor III, whereby R2 to R7 are asdefined as in formula I, nor III and R1 is

Specific compounds of the invention relate to those selected from thegroup comprising of FLI-06, FLI-24, FLI-25, FLI-26, FLI-27 and/or FLI-28as inhibitors of the notch signalling pathway for use as a medicamentfor the treatment of a secretion-dependent disease.

In a preferred embodiment of the invention the medical use for thetreatment of secretion dependent diseases is further defined in thatsecretion-dependent diseases may be selected from those diseases forwhich their aetiology, pathogenesis, morphologic features and/orclinical manifestations are characterised by a dependence on cellularsecretion and/or a secretory pathway, and/or by levels of secretion ofsecreted compounds, preferably proteins, above normal levels (forexample in comparison to a healthy subject or relevant control), wherebya therapeutic effect in the treatment of said diseases is induced by theinhibition of cellular secretion. Relevant controls may relate toindividuals that do not exhibit disease, or exhibit some secretion ofproteins or other substances at a level that does not contribute orcontributes to a smaller extent to a disease state.

The compounds as described herein disrupt a secretory pathway, inparticular the ER and/or golgi apparatus. This represents a beneficialtechnical effect that is common to the compounds disclosed herein andrepresents a surprising effect, neither suggested nor disclosed in theprior art for Notch pathway inhibitors.

The various medical uses encompassed by the invention are thereforeunified by a common functional feature relating to an unexpectedtechnical effect. The role that FLI-06 and derivatives thereof asdescribed herein play in disrupting secretion had been neither disclosednor suggested in the art. A number of diseases rely on secretion withregard to their etiology, pathogenesis, morphologic features and/orclinical manifestations.

Such diseases include, but are not limited to several cancers that aredependent on hyperactive wnt-signaling, a secreted molecule (Herr etal., Trends in Mol. Med. 2012); glioblastoma (Formolo et al. 2011, J.Proteome Research; cancer and inflammatory diseases that secrete variousinterleukins (McLaughlin et al. 2010, British J. of Pharmacology.Inhibition of secretion as anti-cancer drug was suggested for example inOhashi et al. JBC 2011.

A skilled person is able to determine such secretion-dependent diseaseson the basis of existing methods, especially in light of the presentinvention. The experimental examples of the present invention encompassa number of appropriate methods for assaying secretion in vivo or invitro. Such approaches may be applied for example to patient samples inorder to determine the existence of such a disease.

A secretion dependent disease may for example be identified by thedetection of enzymatic reactions in case of enzymatically activesecreted proteins such as disclosed in the SEAP assay, or by measuringthe secreted proteins via ELISA or similar methods known to the skilledperson.

In further embodiments of the invention, the secretion or secretorypathway is related to, mediated and/or characterised by cytokinesecretion, TNFα secretion, IL-6 secretion, IL-8 secretion, IL-10secretion, Wnt secretion, microRNA secretion, CCL2-secretion, ERtransport and/or the Golgi apparatus.

The technical effect of inhibiting membrane traffic is significant forapplication of the compounds of the present invention. Knowledge of thisparticular mechanism provides insight into the requirements necessaryfor appropriate pharmaceutical administration (ie administrationconditions should preferably enable cell entry, compounds could beprepared potentially to be lipid (or organic solvent) soluble), andthereby providing novel information on targeting the disease to betreated.

In one embodiment the information relating to inhibition of membranetrafficking, or disruption of the secretory pathway, preferably bydisruption of the ER and/or golgi apparatus, may lead to treatment ofnovel patient collectives or novel administration regimes, that were notpossible without the information regarding the technical effect and/ormechanism of the molecules described herein.

One aspect of the invention relates to treatment of thesecretion-dependent disease of cancer. The medical use of the compoundsdescribed herein and methods of treatment therefore relate to thetreatment of cancer.

One aspect of the invention relates to treatment of thesecretion-dependent disease of senescence-related ageing. The medicaluse of the compounds described herein and methods of treatment thereforerelate to the treatment of senescence-related ageing and associatedillnesses.

One embodiment of the invention is characterised in that the cancer tobe treated is characterised by a dependency on membrane traffic,secretion or a secretory pathway, preferably related to and/or mediatedby wnt secretion, microRNA secretion, CCL2-secretion, ER transportand/or the Golgi apparatus. In a preferred embodiment the cancersdependent on secretion are selected from chronic lymphocytic leukemia(CLL), esophageal cancer, glioma, colon cancer or breast cancer.

Some cancers are known that are dependent on mechanisms of secretion.For example, a non-comprehensive list of cancers dependent on secretionrelates to chronic lymphocytic leukemia (CLL) (ER transport; Carew, J.S., et al. (2006), Targeting endoplasmic reticulum protein transport: anovel strategy to kill malignant B cells and overcome fludarabineresistance in CLL. Blood 107, 222-231), esophageal cancer (wntsecretion; Fu, L., et al. (2011), Wnt2 secreted by tumour fibroblastspromotes tumour progression in oesophageal cancer by activation of theWnt/beta-catenin signalling pathway. Gut 60, 1635-1643), glioma (wntsecretion; Augustin, I. et al. (2012), The Wnt secretion proteinEvi/Gpr177 promotes glioma tumourigenesis. EMBO Molecular Medicine 4,38-51), colon cancer (wnt secretion), breast cancer metastasis (microRNAsecretion; Breast Cancer-secreted MicroRNAs in the Pre-metastatic Niche,and CCL2 secretion; Loss of TGF-beta signaling in mammary fibroblastsenhances CCL2 secretion to promote mammary tumor progression throughmacrophage dependent and independent mechanisms Stacey L Hembruff, etal. Neoplasia 2010, Volume 12, Issue 5). Because the compounds providedherein show the surprising effect of disrupting secretion pathways at anearly stage in addition to Notch signalling, the invention relates tothe novel combination of notch signalling disruption, secretiondisruption at an early stage and cancer treatment.

One embodiment of the invention is characterised in that the cancer tobe treated is characterised by misregulation of the notch signallingpathway. Preferred cancers are haematological cancer, colorectal cancer,cervical cancer, pancreatic cancer, breast cancer or lung cancer. In apreferred embodiment the haematological cancer is a lymphoma orleukemia. In a preferred embodiment the lymphoma is a T-cell lymphoma,B-cell lymphoma or Hodgkin lymphoma or CLL (Rosati et al, Blood 2009113:856-865).

In a preferred embodiment the compound of the present invention is aninhibitor of the notch signalling pathway intended for use as amedicament in the treatment of cancer, wherein the compound does notdirectly inhibit γ-secretase.

In a preferred embodiment the compound is characterised in that the sideeffects that arise during treatment with the compound are absent orpresent to a lesser extent in comparison to treatment with a directγ-secretase inhibitor. Gamma-secretase inhibitors are well-known asNotch signalling inhibitors, but there are however plagued by variousside effects. The fact the compounds of the present inventiondemonstrate a gamma-secretase independent effect represents a surprisingand beneficial effect, which provides improved conditions for medicalapplication due to the inherently reduced side effects likely to ariseduring treatment due to not directly disrupting gamma-secretase ascurrent notch inhibitors do. An effect of this is that gastrointestinalside effects, such as nausea, diarrhea, vomiting, weight loss and/orloss of appetite are avoided or reduced.

Until the present time, notch signalling inhibitors that do not directlyinfluence gamma-secretase are rare. Those few molecules known thatinhibit notch signalling, but that do not inhibit gamma-secretasedirectly, are not known in cancer treatment, or are not known asdisruptors of membrane transport, and particularly not as disruptors ofER transport or the Golgi apparatus. This therefore represents a noveland non-obvious medical effect. Due to the common feature of non- orindirect gamma-secretase activity (ie the effect against notchsignalling does not directly occur on gamma-secretase), in combinationwith an anti-secretion effect, the compounds of the invention offer thelikely advantage that those side effects associated with GSIs aresignificantly reduced during treatment with the compounds of the presentinvention.

The combination of non-GSI compounds (non-gamma secretase inhibitors;compounds that do not directly influence gamma secretase function),notch signalling inhibition and inhibition of secretion is a novel andnon-obvious development in the field. This combination of compound,mechanism and medical use has not been suggested previously with regardto any of the previously known notch inhibitors. This combination (whichis common to the compounds of the present invention) thereforerepresents a unity-justifying feature, providing a unique contributionto the prior art.

In one embodiment the information relating to non-GSI effect leads totreatment of novel patient collectives or novel administration regimes,that were not possible without the information regarding the technicaleffect and/or mechanism of the molecules described herein. For example,patients who are particularly sensitive to gamma-secretase inhibitorsduring treatment can now also enjoy the benefits of notchinhibition-based treatment due to avoidance of direct gamma secretaseinhibition.

With regard to the treatment of senescence-related ageing and associatedillnesses, a preferred embodiment relates to senescence-related ageingthat is characterised by the presence of senescence-associated secretoryphenotype (SASP).

In one embodiment of the invention the SASP is an inflammatory disease.Such SASP inflammatory disease can in further embodiments of theinvention be induced by secretion of proinflammatory cytokines, inparticular IL-6 and IL-8. Measurements of SEAP-activity in culturemedium or the use of ELISA tests for detecting cytokines, for exampleIL-6 and/or IL-8, may be applied to determine the presence of SASPinflammatory disease.

The invention relates to a pharmaceutical composition comprising one ormore of the compounds disclosed herein and a pharmaceutically acceptablecarrier substance.

The invention therefore also relates to a method for the treatment of asecretion-dependent disease, preferably cancer or senescence-relatedageing, more preferably haematological cancer, colorectal cancer,cervical cancer, pancreatic cancer, breast cancer or lung cancer,chronic lymphocytic leukemia (CLL), esophageal cancer, glioma or coloncancer, more preferably lymphoma or leukemia, such as T-cell lymphoma,B-cell lymphoma, Hodgkin lymphoma or inflammatory disease, comprisingadministration of an effective amount of a pharmaceutical compositioncomprising one ore more of the compounds described herein to a subjectin need of treatment.

Another aspect of the invention relates to the use of one or more of thecompounds of the present invention as a lead structure forderivatization in the development of further biologically activecompounds. Obvious derivatives of the compounds of the presentinvention, developed via further derivatization, are within the skilledpersons reach and fall under the scope of the invention.

Additionally, the present invention relates to the in vitro use of thecompounds described herein as inhibitors of the notch signallingpathway, as inhibitors of secretion or a secretion pathway and/or as aninhibitor of membrane traffic.

In a preferred embodiment the compound as described herein ischaracterised in that the compound has a molecular weight in the rangeof from 100 to 600 g/mol, preferably from 350 to 550 g/mol, a partitioncoefficient of log P≦6, preferably from ≧2 to ≦6, with a maximum of 2hydrogen bridge donors and a maximum of 8 hydrogen bridge acceptors. Thecompounds identified through the screening process described herein allexhibit a variety of common physicochemical properties which make thecompounds highly useful as pharmaceutical agents because they complywith Lipinski's Rule (the so-called Rule of Five) to a very largeextent. This represents a significant advantage in medical applicationof the compounds, which was not expected in light of the prior art. Thesum of structural common features results in a functional relationshipthat provides a common solution to the technical problem of theinvention, namely the provision of notch inhibitors for the treatment ofsecretion dependent diseases that exhibit fewer side effects than themolecules previously known and/or applied in the art. The commonphysicochemical features therefore do not represent an arbitrary sum offeatures, but represent a common fingerprint of the claimed compounds,which advantageously enables and characterizes good in vivo suitabilityof the compounds.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to chemical compounds as inhibitors of the notchsignalling pathway and secretion for the treatment ofsecretion-dependent disease, in addition to pharmaceutical compositionsthereof and methods of treatment. “Secretion-dependent disease” relatesto any disease where inhibition of secretion provides a therapeuticeffect. Preferably, secretion-dependent diseases are selected from thosediseases for which their etiology, pathogenesis, morphologic featuresand/or clinical manifestations are characterised by a dependence oncellular secretion and/or a secretory pathway, whereby a therapeuticeffect in the treatment of said diseases is induced by the inhibition ofcellular secretion. Secretion or a secretory pathway may be understoodas any biological signalling pathway or mechanism involved in cellularsecretion, preferably wherein the secretion or secretory pathway isrelated to, mediated and/or characterised by Wnt secretion, microRNAsecretion, CCL2-secretion, ER transport and/or the Golgi apparatus.

The invention relates to chemical compounds for notch signalling pathwayinhibition for the treatment of cancer or cancer-like disorders, such asa cell proliferative disorder. The terms “cancer”, “cell proliferativedisorder” or “cellular proliferative disorder” are used interchangeablyand refer to any disorder in which the proliferative capabilities of theaffected cells is different from the normal proliferative capabilitiesof unaffected cells. An example of a cell proliferative disorder isneoplasia. Malignant cells develop as a result of a multistep process.The term “malignant” may refer to a tumor or hematopoietic disease nolonger under normal cellular growth control. The term “cancerous cell”as provided herein, includes a cell afflicted by any one of thecancerous conditions provided herein. The term “carcinoma” refers to amalignant new growth made up of epithelial cells tending to infiltratesurrounding tissues, and to give rise to metastases.

A cell proliferative disorder as described herein may be a neoplasm.Such neoplasms are either benign or malignant. The term “neoplasm”refers to a new, abnormal growth of cells or a growth of abnormal cellsthat reproduce faster than normal. A neoplasm creates an unstructuredmass (a tumor) which can be either benign or malignant. The term“benign” refers to a tumour that is noncancerous, e.g. its cells do notproliferate or invade surrounding tissues.

The invention relates to chemical compounds as inhibitors of the notchsignalling pathway and secretion for the treatment of senescence-relatedageing, in addition to pharmaceutical compositions thereof and methodsof treatment. “Senescence-related ageing” refers to senescence, meaninggenerally “to grow old”, or “ageing”. Biological aging is the process ofaccumulative changes to molecular and cellular structure that disruptsmetabolism with the passage of time, resulting in deterioration anddeath. Senescence occurs both on the level of the whole organism(organismal senescence) as well as on the level of its individual cells(cellular senescence). The treatment of senescence is one aspect of thepresent invention and relates to slowing, reversing and/or inhibitingthe ageing process from occurring. During aging the incidence of acuteand chronic conditions such as neurological disorders, diabetes,degenerative arthritis, and even cancer rises within individuals, sothat aging has been termed the substrate on which age-associateddiseases grow. The invention therefore relates to prophylactic methodsfor preventing these diseases by reducing ageing through secretioninhibition.

The molecular pathways underlying aging are not well understood as largeindividual heterogeneity of the biological aging process is observed.These inter-individual differences are proposed to derive fromaccumulation of stochastic damage that is counteracted by geneticallyencoded and environmentally regulated repair systems. At the level ofmolecules repair works by enzymatic systems while on the cellular levelit works by replication and differentiation to maintain tissuehomeostasis. However, the replicative potential of somatic and adultstem cells is limited by cellular senescence and recent evidence showsthat counteracting senescence or removing senescent cells delays theonset of age-associated pathologies. The present invention thereforeprovides means for the treatment of ageing as such, in addition toage-related medical conditions.

In particular, the compounds of the present invention may be applied inthe treatment of inflammation related diseases caused by thesenescence-associated secretory phenotype (SASP). SASP has been reviewedin the literature and the diseases associated with said conditions areknown to a skilled person (see for example Davalos et al., CancerMetastasis Rev. 2010 June; 29(2): 273-283). Several lines of evidencesuggest that tumor suppressor mechanisms can be doubled-edged swords.Such mechanisms suppress the development of cancer early in life; butthey can also result in altered tissue structure, organization, andhomeostasis. These tissue changes can drive phenotypes and pathologiesassociated with aging, including, late-life cancer. Cellular senescencecan be induced by potentially oncogenic stimuli. The senescence responseoften depends on two potent tumor suppressor pathways: that governed bythe p53 protein and that governed by the pRB and p16INK4a proteins.Senescent cells can be found in pre-malignant lesions in mice andhumans, and in mouse models, the senescence response prevents malignantprogression. Consistent with a role in aging, senescent cells accumulatewith age in many rodent, non-human primate, and human tissues. Moreover,they are found at sites of age-related pathology, including degenerativedisorders such as osteoarthritis and atherosclerosis andhyperproliferative lesions such as benign prostatic hyperplasia andmelanocytic naevi. Cell culture and mouse xenograft studies support theidea that senescent cells secrete factors that can disrupt tissuestructure and function and promote cancer progression. Importantly, theSASP may be the major reason for the deleterious side of the senescenceresponse.

Cellular senescence is accompanied by a striking increase in thesecreted levels of >40 factors involved in intercellular signalling. TheSASP has many of the paracrine effects one would expect from apro-inflammatory stimulus, which can be deleterious if left unchecked.The SASP includes several families of soluble and insoluble factors.These factors can affect surrounding cells by activating various cellsurface receptors and corresponding signal transduction pathways thatmay lead to multiple pathologies, including cancer. SASP factors canglobally be divided into the following major categories: solublesignaling factors (interleukins, chemokines, and growth factors),secreted proteases, and secreted insoluble components. SASP proteasescan have three major effects: shedding of membrane-associated proteinsresulting in soluble versions of membrane-bound receptors,cleavage/degradation of signaling molecules, and degradation orprocessing of the extracellular matrix.

The terms “γ-secretase” and gamma-secretase are used interchangeably.

In the present invention “treatment” or “therapy” generally means toobtain a desired pharmacological effect and/or physiological effect. Theeffect may be prophylactic in view of completely or partially preventinga disease and/or a symptom, or may be therapeutic in view of partiallyor completely curing a disease and/or adverse effect of the disease. Inthe present specification, “therapy” includes arbitrary treatments ofdiseases or conditions in mammals, in particular, humans, for example,the following treatments (a) to (c): (a) Prevention of onset of adisease, condition or symptom in a patient; (b) Inhibition of a symptomof a condition, that is, prevention of progression of the symptom; (c)Amelioration of a symptom of a condition, that is, induction ofregression of the disease or symptom.

The term “pharmaceutical composition” refers to a combination of theagent as described herein with a pharmaceutically acceptable carrier.The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce a severe allergic or similaruntoward reaction when administered to a human. As used herein,“carrier” or “carrier substance” includes any and all solvents,dispersion media, vehicles, coatings, diluents, antibacterial andantifungal agents, isotonic and absorption delaying agents, buffers,carrier solutions, suspensions, colloids, and the like. The use of suchmedia and agents for pharmaceutical active substances is well known inthe art. Supplementary active ingredients can also be incorporated intothe compositions. A pharmaceutical composition of the present inventioncan include pharmaceutically acceptable salts of the components therein.The pharmaceutical composition containing the active ingredient may bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions and suchcompositions. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients which are suitable forthe manufacture of tablets. The tablets may be uncoated or they may becoated by known techniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period.

Dosage levels of the order of from about 0.01 mg to about 500 mg perkilogram of body weight per day are useful in the treatment of theindicated conditions. For example, a cancer may be effectively treatedby the administration of from about 0.01 to 50 mg of the inventivemolecule per kilogram of body weight per day (about 0.5 mg to about 3.5g per patient per day). The amount of active ingredient that may becombined with the carrier materials to produce a single dosage form willvary depending upon the host treated and the particular mode ofadministration. For example, a formulation intended for the oraladministration of humans may vary from about 5 to about 95% of the totalcomposition. Dosage unit forms will generally contain between from about1 mg to about 500 mg of active ingredient. It will be understood,however, that the specific dose level for any particular patient willdepend upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet timeof administration, route of administration, rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy. The dosage effective amount of compounds according to theinvention will vary depending upon factors including the particularcompound, toxicity, and inhibitory activity, the condition treated, andwhether the compound is administered alone or with other therapies.Typically a dosage effective amount will range from about 0.0001 mg/kgto 1500 mg/kg, more preferably 1 to 1000 mg/kg, more preferably fromabout 1 to 150 mg/kg of body weight, and most preferably about 50 to 100mg/kg of body weight. The invention relates also to a process or amethod for the treatment of the mentioned pathological conditions. Thecompounds of the present invention can be administered prophylacticallyor therapeutically, preferably in an amount that is effective againstthe mentioned disorders, to a warm-blooded animal, for example a human,requiring such treatment, the compounds preferably being used in theform of pharmaceutical compositions.

In one aspect, the invention provides a method of preventing, treating,and/or managing cancer to a patient in need thereof, the methodcomprising administering a prophylactically effective regimen or atherapeutically effective regimen, the regimen comprising administeringto the patient a compound of the invention, or a pharmaceuticallyacceptable salt thereof, wherein the patient has been diagnosed withcancer, and wherein said cancer is a haematological cancer, colorectalcancer, cervical cancer, pancreatic cancer, breast cancer or lungcancer. In some embodiments, a patient may receive therapy for thetreatment and/or management of the cancer before the administration ofthe therapeutically effective regimen of the compound of the invention,or a pharmaceutically acceptable salt thereof. Non-limiting examples ofsuch a therapy include chemotherapy, radioimmunotherapy, toxin therapy,prodrug-activating enzyme therapy, antibody therapy, surgical therapy,immunotherapy, radiation therapy, targeted therapy (i.e. therapydirected toward a specific target or pathway, e.g. tyrosine kinase,etc.), and any combination thereof. In some embodiments, the patient hasnot previously received a therapy for the treatment and/or management ofthe cancer. In a specific embodiment, the hematologic cancer is lymphomaor leukemia, or a T-cell lymphoma, B-cell lymphoma or Hodgkin lymphoma.

In another aspect, the invention provides a method of preventing,treating, and/or managing a solid tumor in a patient, the methodcomprising administering to a patient in need thereof a prophylacticallyeffective regimen or a therapeutically effective regimen, the regimencomprising administering to the patient a compound of the invention, ora pharmaceutically acceptable salt thereof, wherein the patient has beendiagnosed with a solid tumor. In particular embodiments of this aspect,the solid tumor is fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon cancer, colorectal cancer, kidney-cancer,pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostatecancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer,throat cancer, squamous cell carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicularcancer, small cell lung carcinoma, bladder carcinoma, lung cancer,epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skincancer, melanoma, neuroblastoma, or retinoblastoma.

The invention is therefore based on a novel type of secretion inhibitor,FLI-06 and derivatives thereof. In contrast to other secretioninhibitors, no gross ER stress is induced. FLI-06 and its derivativesrepresent useful medicaments for inhibiting or reducing secretionwithout inducing ER-stress mediated apoptosis. The following analyseshave been conducted or are appropriate for further analysis of FLI-06and its derivatives, in order to identify and support the effect ofthese compounds in interfering with SASP to delay tissue dysfunction andextend healthy lifespan:

FLI-06 is a secretion inhibitor with an EC₅₀ of 2.3 μM. We performedinitial structure-activity relationship (SAR) studies that will beextended in the current proposal to develop inhibitors working in thenanomolar range. FLI-06 and derivatives will be tested for theirefficacy in human fibroblasts where senescence is induced chemically orby repetitive passaging. Inhibition of SASP can be measured with ELISAsfor IL-6 and -8 and by measuring secreted alkaline phosphatase (SEAP)(Gu, L. & Kitamura, M. Sensitive detection and monitoring ofsenescence-associated secretory phenotype by SASP-RAP assay. PLoS One 7,e52305, (2012)). Cellular toxicity, proliferation as well as autophagycan be assayed. To study the role of SASP in brain ageing, promisingcompounds can be analyzed whether they can reverse defects in learningand memory in brain slices of ageing models like the Klotho mouse andwhether dynamics of UPS and lifetime of synaptic proteins are changed.Promising compounds can also be tested for life-extending effects in C.elegans. Lead substances are being further optimized for use invertebrate animal trials.

Novel SASP inhibitors such as those described herein can also further beassayed or screened with senescent human fibroblasts that stably expressSEAP. Hits can be validated in secondary screens involving serialdilutions and IC₅₀ determinations. Hits can be tested whether theyaffect general secretion in non-senescent and senescent cells. Promisinghits can be analyzed for their mode of action, specificity and toxicity.

Additionally, genome-wide siRNA screens can be carried out to identifynovel targets involved in senescence, SASP and autophagy. Multi-colorhuman fibroblast cell lines have been generated that stably expressesfluorescent-protein tagged markers for the Golgi (GaIT, the Golgi isenlarged and dispersed in senescence), lysosomes (Lamp1, lysosomes areenlarged in number and size in senescence) and autophagosomes (LC3,number is increased in senescence). For automatic identification ofcells, the nuclei can be labeled with the far-red nuclear dye To-Pro-3(Invitrogen). Conditions may be optimized for transfection and screeningof chemically-induced senescent multi-color cells in 384-well format.Read-out parameters can include grade of dispersal of the Golgi, numberand size of lysosomes, number and size of autophagosomes. Genome-widesiRNA libraries (Dharmacon) can additionally be screened, andphenotypical changes in the three markers analyzed. Hits can beclassified: Hits affecting the senescent secretory pathway on alllevels, hits affecting only one or two of the read-out parameters, hitsaffecting autophagy with or without affecting Golgi or lysosomes, andmore. Candidates validated in secondary screens can be tested in detail,for example whether the morphological change they induce indeed affectsSASP, whether it is causative or correlative for SASP and/or senescence,whether the affected gene was associated to ageing before.

The experimental methods described above can therefore be used toprovide further information on the derivatives disclosed herein andrelated compounds falling under the general formulas disclosed herein.Furthermore, additional compounds and genetic factors involved insenescence and the mechanism of action of the compounds as describedherein may be identified by the experimental approaches describedherein.

FIGURES

The figures provided herein represent examples of particular embodimentsof the invention and are not intended to limit the scope of theinvention. The figures are to be considered as providing a furtherdescription of possible and potentially preferred embodiments thatenhance the technical support of one or more non-limiting embodiments.

Short description of the figures:

FIG. 1: Chemical interference of Notch trafficking/processing isamenable to automated microscopy.

FIG. 2: Selected compounds from final hit list show distinct phenotypes.

FIG. 3: Effect of compounds on NotchΔE-EGFP, APP and Klotho traffickingand processing.

FIG. 4: Selected compounds inhibit endogenous Notch signaling.

FIG. 5: The dihydropyridine FLI-06 disrupts the Golgi by mechanismsdifferent from BFA or GCA.

FIG. 6: FLI-06 affects recruitment of cargo to ER exit sites.

FIG. 7: FLI-06 induces ER sheet formation.

FIG. 8: DAPT-like phenotype of FLI-14, -15, -19 and -20 at 50 μM.

FIG. 9: Compound effects are not cell-type specific.

FIG. 10: Phenotypes of Notch inhibitors are fully reversible.

FIG. 11: Selected compounds affect somite formation and neurogenesis invivo in zebrafish embryos.

FIG. 12: FLI-06 disrupts the Golgi not via depolymerizing microtubuli oractin.

FIG. 13: FLI-06 causes dispersal of early Golgi and TGN, but notubulation of endosomes.

FIG. 14: FLI-06 acts different from BFA.

FIG. 15: FLI-06 inhibits transport of GPI-anchored and soluble proteinsand does not cause ER stress.

FIG. 16: Only ER exit inhibitors convert ER tubules to sheets.

FIG. 17: FLI-06 blocks ER-export instantaneously.

FIG. 18: FLI-06 inhibits secretion of secreted alkaline phosphates;FLI-06 kills cancerous, Notch dependent T-cells

FIG. 19: Molecular structure of FLI-06. Thermal Ellipsoids are displayedat 40% probability level.

Detailed description of the figures:

FIG. 1: Chemical interference of Notch trafficking/processing isamenable to automated microscopy. Scheme of the membrane bound reporterNotchΔE-EGFP (a) and its cellular trafficking (b). NotchΔE-EGFP issynthesized in the ER and transported through the Golgi to the plasmamembrane (PM). At the PM γ-secretase cleavage releases NICD-EGFP, whichis then translocated to the nucleus. c) Scheme of automated imageacquisition and quantitation. Fixed HeLa-NotchΔE-EGFP cells were stainedwith DAPI and imaged by automated microscopy. A nuclear mask and a ringaround the nucleus was created and used for determination of nuc, enucand enuc/nuc ratio of fluorescence intensities. d) HeLa-NotchΔE-EGFPcells were incubated with or without DAPT, fixed and imaged byfluorescence microscopy. e) Quantitation of DAPT dose-dependent enucaccumulation of NotchΔE-EGFP and time-dependent reduction of nuclearNICD-EGFP after DAPT treatment. f, g) HeLa-NotchΔE-EGFP cells wereincubated with increasing amounts of monensin, fixed after 6 h andanalyzed by automated microscopy. f) Example image of cells treated withDMSO or monensin (5 μM). Accumulated fluorescence in the Golgi is markedby arrows. g) Quantitation of >100 cells±SE. Displayed is the nuc/enucratio. h, i) HeLa-NotchΔE-EGFP cells were incubated in the presence orabsence of MG132 (1 μM) for 16 h, fixed and processed for fluorescencemicroscopy (h). i) Quantitation of nuclear fluorescence of >250cells±SE. All y-axes display fluorescence intensity in arbitrary units.Scalebar 10 μm. j) Summary of the screen.

FIG. 2: Selected compounds from final hit list show distinct phenotypes.Hela NotchΔE-EGFP cells were incubated with compounds (10 μM) for 18 h,fixed and analyzed by fluorescence microscopy (a) or lysed, separated bySDS-PAGE, blotted and probed with antibodies against GFP and NICD (b).Tubulin served as loading control. c) Quantitation of NotchΔE-EGFPaccumulation and NICD production from 3-4 experiments as shown in (b).Displayed is the SD with asterisks marking p<0.05. Arrows, plasmamembrane; scalebar, 10 μm.

FIG. 3: Effect of compounds on NotchΔE-EGFP, APP and Klotho traffickingand processing. a) HEK293 stably expressing APP_(swe) or Klotho (KI)were incubated with DAPT or compounds (10 μM) for 16 h. Media werecollected, cells lysed and both assayed by Western blotting usingindicated antibodies. m, mature; im, immature; s, shedded ectodomain;CTF, C-terminal fragment. b) Membranes from HeLa-NotchΔE-EGFP and APPswecells were isolated and subjected to a γ-secretase in vitro assay in thepresence of DAPT or compounds as indicated. NICD and AICD were detectedby Western Blotting using antibodies against the C-terminus of APP (top)or antibodies specific for NICD (bottom). c) Quantitation from 5independent experiments. Displayed is the SD with asterisks markingp<0.01.

FIG. 4: Selected compounds inhibit endogenous Notch signaling. a) C2C12cells were transfected with the Notch ligand Delta and aluciferase-based Notch-reporter. Cells were incubated with DAPT or 10 μMof the indicated compounds (50 μM in case of FLI-15 and -20) and after16 h a luciferase assay was performed. Values from DMSO-treated cellswere set to 0% inhibition, from DAFT-treated cells to 100% inhibitionand the other compounds related to that. b) and c) Compounds (50 μM)were added to dechorionated stage 4 hpf zebrafish embryos and theeffects analyzed after 24 h. b) qPCR with primers specific for ngn1.Changes in relative gene expression levels (fold change) relative to theexpression levels in DMSO controls are represented as the mean+/−SD,asterisks indicate p<0.01. Each measurement was taken from twoindependent samples. Each reaction was measured in triplicate. Changesin the relative expression of ngn1 were standardized to the expressionof the housekeeping gene ef1a at 20 hpf. c) ISH with riboprobes specificfor ngn1 was performed on dechorionated stage 4 hpf zebrafish embryos.Anterior is to the left, dorsal to the top. Magnifications of the somiteregion (small box) are shown. Arrows indicate larger ngn1 clusters orstronger ngn1 staining. s, sensory neurons; i, intermediate neurons; m,motor neurons.

FIG. 5: The dihydropyridine FLI-06 disrupts the Golgi by mechanismsdifferent from BFA or GCA. a) For structure-activity relationshipsFLI-06 (1) and derivatives (2 to 7) were incubated on HeLa-NotchΔE-EGFPcells and EC₅₀ values determined. b) Results from testing compoundsusing the HeLa-NotchΔE-EGFP cells. c) Structural information on thecompounds tested according to the experiment shown in b). d) HeLa cellswere incubated with DMSO or FLI-06 (10 μM) for 18 h, fixed, stained withantibodies as indicated and imaged by fluorescence microscopy. e) HeLacells were transfected with GBF1-GFP, incubated the next day with DMSOor FLI-06 (10 μM) or BFA (10 μg/ml) or GCA (10 μM) for 10 h, fixed, andimaged by fluorescence microscopy. Scalebar: 10 μm.

FIG. 6: FLI-06 affects recruitment of cargo to ER exit sites. a, a′, b)HeLa cells were transiently transfected with VSVG-EGFP and incubatedovernight at 40° C. 30 min before the chase microtubuli weredepolymerized by incubation on ice and treatment with nocodazole.Concurrently, cells were preincubated with DMSO (control) or FLI-06 (10μM). Cells were then chased at 32° C. for indicated times in thepresence of nocodazole and compounds, followed by fixation andprocessing for fluorescence microscopy using antibodies against Sec31.a) Overview of treated and untreated cells at selected time points.Scalebar 10 μm. a′) Magnified selected areas from a); V, VSVG-EGFP; S,Sec31; M, merge. Double arrow, VSVG-EGFP in post-ERES compartment, nextto an ERES; arrowhead, ERES costained with VSVG-EGFP and Sec31; scalebar3 μm. b) Quantitation of variance of pixel fluorescence intensity(PFI-Var) in region of interests (ROI) from n=3 experiments as in a).For each condition at least 10 ROIs were measured. Error bars SEM. r.u.,relative units. Mean PFI-Var at time 0 was set to 1 and the other valuesrelated to that. c) HeLa-NotchΔE-EGFP cells were pretreated for 4 h withFLI-25, an inactive derivative of FLI-06 (see FIG. 5), or with FLI-06where indicated. Cells were permeabilized and the budding reactionperformed with cytosol, ATP regenerating system and compounds asindicated. After the reaction COPII vesicles were isolated and probedfor ribophorin I (ER marker), ERGIC-53 and Sec22b (both proteins areincorporated in COPII vesicles).

FIG. 7: FLI-06 induces ER sheet formation. A) COS cells transfected withprlss-KDEL-mRFP were incubated with 10 μM FLI-06 for indicated times andimaged by live-cell microscopy. The inserts are magnifications of theboxed areas. B) Images from cells treated as in A) were scored for thepercentage of cells that lost most of their ER-tubules. For each timepoint 40-60 cells were counted. Data represent mean percentages, errorbars SEM, n=3 independent experiments.

FIG. 8: DAPT-like phenotype of FLI-14, -15, -19 and -20 at 50 iM. HeLacells stably expressing NotchΔE-EGFP were incubated with 1 iM DAPT or 50iM compounds for 16 h, fixed and processed for immunofluorescence. Inall conditions, cells displayed a strong PM accumulation of NotchΔE-EGFPand a strong reduction in nuclear NICD-EGFP.

FIG. 9: Compound effects are not cell-type specific. U2OS cells stablyexpressing NotchΔE-EGFP were incubated for 18 h with 10 iM compounds asindicated, fixed and imaged by fluorescence microscopy. FLI-14, -15, -19and -20 cause PM accumulation similar to DAPT, FLI-06 caused ERaccumulation of the reporter and in some cells aggregation, indicatingthat phenotypes of all compounds are not cell-type specific. Arrows,plasma membrane; arrowheads, ER. Asterisks indicate cells where the ERcollapsed to large aggregates. Scalebar, 10 im.

FIG. 10: Phenotypes of Notch inhibitors are fully reversible. HeLaNotchΔE-EGFP cells plated in LabTek coverglass were incubated for 18 hwith 10 iM of indicated compounds, washed and incubated for indicatedtimes in cell culture medium. Cells were imaged by live-microscopy. ERaccumulation (FLI-06) or PM accumulation (FLI-14, -15, -19, -20) ofNotchΔE-EGFP reversed after 1-4 h of washout to the normal accumulationof NICD-EGFP in the nucleus, demonstrating full reversibility ofcompound effects. In addition these data show no gross toxicity of thecompounds. Scalebar, 10 im.

FIG. 11: Selected compounds affect somite formation and neurogenesis invivo in zebrafish embryos. a) Lateral view of typical representativephenotypes induced by DAPT treatment, which were used as a reference toscore the effect of compounds. Embryos treated at 6 hpf with 50 iM DAPTresulted in either defective somitogenesis (mild), head and trunkmalformation (strong) or developmental delay (delayed). Images weretaken at 24 hpf. b) Pie chart showing the cumulative relative frequencyof the phenotypes in a) at 24 hpf in embryos treated with 20, 50 and 100iM of the indicated compounds. c) ISH with riboprobes specific for ngn1was performed on dechorionated stage 4 hpf zebrafish embryos. Anterioris to the left, dorsal to the top. Magnifications of the developing head(box) are shown. Arrows indicate larger ngn1 clusters or stronger ngn1staining. t, telencephalon; p, protectum; h, hindbrain; d, diencephalon;tg, tegmentum; vrc, ventral rostral cluster.

FIG. 12: FLI-06 disrupts the Golgi not via depolymerizing microtubuli oractin. HeLa cells were incubated for 4 h with 1.5 ig/ml nocodazole (a)or 2 iM cytochalasin D (b) or 10 iM FLI-06. Thereafter cells were fixedand processed for immunofluorescence microscopy using antibodies againsttubulin. a) Nocodazole, but not FLI-06 disrupted the microtubularnetwork in interphase cells and the mitotic spindle (arrowheads) inmitosis. b) Cytochalasin D, but not FLI-06 depolymerized f-actin.Scalebar, 10 im.

FIG. 13: FLI-06 causes dispersal of early Golgi and TGN, but notubulation of endosomes. Hela cells were incubated with DMSO or 10 iMFLI-06 or 1 ig/ml BFA for 18 h, fixed, stained with antibodies asindicated and imaged by fluorescence microscopy. Endosomes werevisualized by adding Alexa555-labelled transferrin on ice and chasingfor 15 min at 37° C. in the presence of indicated compounds. Arrowsindicated tubulating endosomes, scalebar 10 im.

FIG. 14: FLI-06 acts different from BFA. a) HeLa cells were incubatedfor indicated time points with 1 ig/ml BFA or 10 iM FLI-06, fixed andprocessed for immunofluorescence microscopy using antibodies againstβCOP and giantin. While with BFA βCOP rapidly dissociates from the Golgiwithin 10 min, it takes much longer for FLI-06. b) HeLa cells weretransfected with prlss-KDEL-mRFP (ER) and B4GALT-EGFP (Golgi), incubatedfor indicated time points with 1 ig/ml BFA or 10 iM FLI-06, fixed andprocessed for immunofluorescence microscopy. In contrast to BFA, FLI-06does not lead to fusion of ER and Golgi. The ER-like staining of theGolgi-marker at 360 min is probably due to newly synthesized protein. c)HeLa cells were transfected with VSVG-EGFP and incubated at 40° C.throughout to keep VSVG-EGFP in the ER. Cells were treated for 4 h at40° C. with 10 ig/ml cycloheximide and DMSO, BFA or FLI-06 as indicated,lysed and subjected to SDS PAGE. Incubation with BFA but not FLI-06 ledto hyperglycosylation in the ER, indicated by the lower mobility inSDS-gels (hyperglyc.). This hyperglycosylation suggests thatGolgi-resident enzymes redistributed to the ER in case of BFA, but notFLI-06.

FIG. 15: FLI-06 inhibits transport of GPI-anchored and soluble proteinsand does not cause ER stress. a) HeLa cells were transiently transfectedwith YFP-GPI (kindly provided by Patrik Keller) and incubated for 18 hwith DMSO or 1 ig/ml BFA or 10 iM FLI-06. Thereafter cells were fixedand analyzed by immunofluorescence microscopy. FLI-06 and BFA inhibitsurface transport of YFP-GPI. b) HeLa cells were transiently transfectedwith a plasmid coding for the secreted ectodomain of Klotho, KlothoS,kindly provided by Makoto Kuro-o). After incubating with compounds for18 h media were collected, cells lysed and both analyzed by WesternBlotting using Klotho antibody. As loading control membranes were probedfor actin. BFA and FLI-06 inhibit secretion of KlothoS. c) Hela cellswere incubated with indicated compounds, lysed after 6 or 24 h, blottedand probed for BIP or actin as loading control. d) Hela cellstransfected with the luciferase-based ER-stress indicator plasmidp5xATF6-GL3 (Addgene #11976) and Renilla control plasmid were incubatedwith indicated compounds, lysed after indicated time points andluciferase activity was determined. 21 g/ml Tunicamycin (Tun) and 5 mMDTT were used as positive controls. In both assays FLI-06 caused onlyvery mild ER-stress after 24 h. Shown is the average of threeindependent experiments, error bars indicate standard deviation.

FIG. 16: Only ER exit inhibitors convert ER tubules to sheets. COS cellsplated on LabTek chambered coverslips were transiently transfected withthe ER-marker prlss-KDEL-mRFP, incubated the next day for 2 h with 10 iMof indicated compounds and imaged by live-cell microscopy. Only the ERexit inhibitors (see FIG. 5 a) FLI-06, 3, 4 and 5 showed prominent ERsheet formation. FLI-06+CHX, cells were additionally incubated in 40 iMcycloheximide. Nife, nifedipine; nimo, nimodipine. Numbers refer tonumbering in FIG. 5 a.

FIG. 17: FLI-06 blocks ER-export instantaneously. HeLa cells weretransiently transfected with VSVG-EGFP and incubated overnight at 40° C.30 min before the chase microtubuli were depolymerized by incubation onice and treatment with nocodazole. In addition, cells were preincubatedfor 30 min with DMSO (control) or FLI-06 (FLI-06+30 min pre), or for 10min with FLI-06 (+10 min pre), or FLI-06 was added only to the chasemedium (FLI-06 no pre). Cells were then chased at 32° C. for indicatedtimes in the presence of nocodazole and FLI-06 (except control),followed by fixation. Quantitation of variance of pixel fluorescenceintensity (PFI-Var) was performed in regions of interest (ROI) in atleast 15 cells per condition per experiment. n=3 independentexperiments, error bars SEM. a.u., arbitrary units. Even when added onlyto the chase medium without preincubation FLI-06 inhibits concentrationand subsequent ER-export of VSVG-EGFP.

FIG. 18: FLI-06 inhibits secretion of secreted alkaline phosphates;FLI-06 kills cancerous, Notch dependent T-cells. a) The secretedalkaline phosphatase (SEAP) assay was carried out. Medium was collectedand SEAP secretion was measured via photometry. Inhibition of secretionof SEAP upon FLI-06 incubation was confirmed. b) DND-41 cells haveactivating Notch mutations (Weng et al, 2004, Science 306, 269). Thecells were grown in suspension with the indicated amounts of FLI-06 orDMSO control and growth was determined via FACS analysis. Mediumconcentrations (1 μM) of FLI-06 inhibited proliferation, while highconcentrations (10 μM) of FLI-06 killed the cancer cell line.

FIG. 19: Molecular structure of FLI-06. Thermal Ellipsoids are displayedat 40% probability level.

Examples

The examples provided herein represent practical support for particularembodiments of the invention and are not intended to limit the scope ofthe invention. The examples are to be considered as providing a furtherdescription of possible and potentially preferred embodiments thatdemonstrate the relevant technical working of one or more non-limitingembodiments.

Establishment of a Microscope-Based High-Content Screen

To identify novel regulatory factors involved in Notchtrafficking/processing an image-based high content screen was set up. Tothis end a HeLa Kyoto cell line stably expressing an EGFP-tagged,transcriptionally inactive and ligand-independent Notch1-construct wasemployed (FIG. 1 a, NotchΔE-EGFP¹⁵). NotchΔE-EGFP is a direct substratefor γ-secretase at the PM, and under physiological conditions isproteolytically processed to NICD-EGFP that translocates into thenucleus (for scheme see FIG. 1 b), resulting in a strong nuclearEGFP-staining in steady state¹⁵ (FIG. 1 d). Upon inhibition ofγ-secretase with the GSI DAPT, the reporter accumulated at the PM whilenuclear fluorescence decreased (FIG. 1 d). Importantly, changes insubcellular localization of the reporter are amenable to quantitation byautomated microscopy¹⁵. Accordingly, reporter fluorescence wasdetermined in the nucleus (nuc) and in a ring around the nucleus (enuc),and the ratio enuc/nuc was evaluated (schematized in FIG. 1 c).Thereafter, the assay design was functionally validated. DAFT-inducedaccumulation of the reporter outside the nucleus was foundconcentration-dependent with an EC50 of 0.68±0.2 iM (18 h incubation,FIG. 1 e). NICD-EGFP fluorescence in the nucleus decreased with at_(1/2) of 4.7±1.0 h and t_(g/10) of 8.6±2.9 h at 1 μM DAPT (FIG. 1 e).Visual inspection suggested that the cells were viable at all times. Weshowed before that with these cells defects in nuclear import of NICDcould be quantified by automated microscopy¹⁵. The assay also reliablydetected trafficking defects in the secretory pathway. For example,blocking the anterograde transport of NotchΔE-EGFP through the secretorypathway at the Golgi by the ionophore monensin¹⁶ in a dose-dependentmanner could be quantified by automated microscopy (FIG. 1 f, g).Furthermore, accumulation of NICD in the nucleus upon inhibitingproteasomal degradation by MG132 was detected by automated microscopy(FIG. 1 h, i). Taken together, the assay was validated for reliablydetecting concentration and time dependent changes in trafficking andprocessing of the Notch reporter.

After assay conditions were optimized for 384 well plates and automatedliquid handling, the NotchΔE-EGFP reporter cell line was screenedagainst the ChemBioNet library comprising 16,671 compounds¹⁷ (summarizedin FIG. 1 j). Enuc/nuc ratio was computed and the 352 top-scoringcompounds were investigated in follow-up experiments. Images werevisually inspected for phenotype, and cell viability and EC₅₀ valueswere determined, resulting in a primary hit list of 68 compounds. Fordetailed follow-up studies seven compounds were selected (table 1), noneof which had been annotated with relevant bioactivity before.

Table 1: Top 7 hit list from the compound screen. Images from the 352initial hits were visually inspected and 7 compounds with prominentphenotypes and intact nuclei were selected. EC₅₀ values were determinedas described in the methods section.

Selected Compounds Block Notch Trafficking/Processing at Distinct Steps

For a detailed analysis of subcelluar distribution of NotchΔE/NICD theresolution of the HCS images was not sufficient. Therefore, the reportercell line was plated on coverslips and incubated with 10 μM ofindividual compounds from the final hit list. After 24 h thelocalization of NotchΔE-EGFP/NICD-EGFP was determined by fluorescencemicroscopy. As shown above, in steady state the reporter fluorescencewas localized to the nucleus and accumulated at the PM after DAPTtreatment (FIG. 2 a). Incubation with FLI-14, -15, -19 and -20 caused anaccumulation of the reporter at the PM and in perinuclear Golgi-likestructures similar to DAPT (FIG. 2 a). Nuclear NICD-EGFP fluorescencedid not decrease to the extent observed for DAPT, suggesting thecompounds are less effective than DAPT. Indeed, strong reduction ofnuclear NICD-EGFP fluorescence and concomitant accumulation at the PMwas achieved after incubation with 50 μM of FLI-14, -15, -19 and -20.(FIG. 8). FLI-06 treatment resulted in reduced nuclear fluorescence andlocalization in intracellular membranes. Other compounds did not alterthe reporter localization in this primary validation (FIG. 2 a, FLI-13,FLI-16).

Next, lysates of HeLa NotchΔE-EGFP cells treated with compounds wereanalyzed for processing of NotchΔE-EGFP by Western blot using antibodiesspecific for EGFP and NICD (FIG. 2 b, c). The GFP antibody detected boththe uncleaved reporter NotchΔE-EGFP and the γ-secretase productNICD-EGFP, whereas the NICD-specific antibody specifically detectedcleaved NICD-EGFP. Treatment of cells with FLI-06, -14, -15, -19 and -20resulted in accumulation of NotchΔE-EGFP. Concomitantly, FLI-06, -14,-15 and -20 resulted in decreased NICD-EGFP production. FLI-13 andFLI-16 had no influence on NotchΔE-EGFP processing. The observed effectswere not cell type specific, as they were also apparent in U2OS cellsstably expressing NotchΔE-EGFP (FIG. 9). Live-cell microscopy afterwash-out of the components indicated that phenotypes of FLI-06, -14,-15, -19 and -20 were fully reversible within 1-4 h, which alsoindicates that the compounds are not acutely toxic in cells (FIG. 10).

Four Compounds are γ-Secretase Inhibitors

The accumulation of NotchΔE-EGFP at the PM and the reduction ofNICD-EGFP in the nucleus suggested that FLI-14, -15, -19 and -20affected γ-secretase processing. γ-secretase has many substrates besidesNotch, most prominently the amyloid precursor protein APP, for reviewsee². To test if the compounds that affected NotchΔE-EGFP processingalso affected APP processing, HEK293 cells were used that stablyexpressed APP_(swe), a mutated APP that yields robust amounts of Aβ¹⁸.Aβ in the cell supernatant was weakly reduced after treatment withFLI-15 and -20 but essentially disappeared after treatment with FLI-06and FLI-14, similar to the GSI DAPT (FIG. 3 a). Likewise, APP_(CTF), thedirect substrate of γ-secretase, accumulated in FLI-14, -15 and -20treated cells, consistent with the effects on Aβ, In FLI-06 treatedcells no APP_(CTF) accumulated despite strongly reduced Aβ secretionsuggesting that APP is not cleaved by β-secretase. All other compoundshad no effect on APP_(CTF) or Aβ. When analyzing APP and APPs, theshedded ectodomain of APP processed by α- and β-secretase¹⁹, we observedthat FLI-06, but not the other compounds changed the glycosylationpattern of APP and abolished the shedding of APPs. FLI-16, -19 and -20reduced APPs secretion (FIG. 3 a).

To test whether FLI-06 affected other membrane proteins, Klotho, a typeI protein processed like APP and Notch²⁰, was analyzed. Treatment ofHEK293 cells stably expressing Klotho with FLI-06, but not with theother compounds, resulted in aberrant glycosylation different from thenormal immature and mature forms of Klotho (FIG. 3 a). The mature,complex-glycosylated post-Golgi form of Klotho was strongly reduced,suggesting that FLI-06 affected the trafficking of Klotho. Likewise,when shedded Klotho_(s) derived from α- and β-secretase cleavage wasassayed, FLI-06 but not the other compounds led to a strong reduction ofKlotho_(s) in the medium, suggesting that FLI-06 inhibited cell surfacetransport (FIG. 3 a). These data suggested that FLI-14, -15, -19 and -20acted on γ-secretase, whereas FLI-06 probably acted by interfering withtransport in the secretory pathway. To analyze γ-secretase activity, invitro assays using isolated membranes were performed (FIG. 3 b, c).Production of NICD-EGFP and AICD (APP intracellular domain) wereassayed. Both were produced in vitro at 37° C. but not at 4° C., andtheir production was inhibited by DAPT. Among the tested compounds, onlyFLI-15 and FLI-20 clearly reduced NICD-EGFP and AICD production,suggesting that they acted as direct, non-substrate selectiveγ-secretase inhibitors. In contrast, FLI-06, -14 and -19 did not affectγ-secretase in isolated membranes (FIG. 3 b, c). FLI-13 and -16, shownnot to be active in the cellular assays, also did not affect in vitroγ-secretase activity. Taken together, these data suggest that (i) FLI-15and -20 inhibited γ-secretase in cells and in vitro, (ii) FLI-14 and -19inhibited Notch and APP processing in cells, but not γ-secretase invitro and (iii) FLI-06 affected general transport through the secretorypathway.

Effects of Compounds on Endogenous Notch Signaling

We wanted to test whether the results obtained with the NotchΔE-EGFPconstruct are relevant for endogenous Notch signaling. Therefore we madeuse of C2C12 cells, a well established model for muscle development thatexpresses endogenous Notch^(21,22). After stimulating endogenous Notchsignaling by transfecting the Notch ligand Delta, Notch activity wasmeasured by a luciferase assay using a Notch reporter¹⁵ in the presenceor absence of compounds (FIG. 4 a). FLI-06, -14 and -16 strongly, andFLI-19 considerably inhibited endogenous Notch signaling at 10 μM,similar to DAPT. FLI-15 and -20 hardly affected Notch signaling at 10μM, but remarkably at 50 μM (FIG. 4 a, only 50 μM values are shown).Next, we tested whether selected compounds would affect Notch signalingin vivo. To this end the zebrafish Danio rerio was used because of itsversatility to study early developmental processes and because it showsa distinct Notch phenotype in somitogenesis and neurogenesis whentreated with GSI^(23,24). Compounds were added to dechorionated stage 4hpf zebrafish embryos and the effects were analyzed after 24 h bymorphological inspection of somite formation. Phenotypes were ranked tofour categories, normal somites, mild somite defects, strong somitedefects and developmental delay. All compounds induced distinct Notchphenotypes similar to DAPT (FIG. 11 a, b). As an additional parameter wedecided to analyze Notch-dependent genes involved in neurogenesis.Primary neurogenesis in zebrafish involves the selection of neuroblasts(repressed by Notch) from proneural clusters (Notch expressing cells), aprocess regulated in a negative manner by Notch-dependent lateralinhibition²⁵. Accordingly, neuroblast cells that express the neuronalspecification factor neurogenin (ngn1²⁶) became more abundant when Notchsignaling was impaired, for instance upon DAPT treatment^(23,27). qPCRanalyses on mRNA extracted from equivalent sets of embryos was performedfor ngn1 expression (FIG. 4 b). Upon treatment with DAPT, FLI-06, -14,-15 and -20 ngn1 mRNA levels were significantly increased, whiletreatment with FLI-16 and -19 did not induce significant changes in ngn1mRNA levels. To confirm the data obtained by qPCR, whole-mount in-situhybridizations (ISH) using an ngn1 specific riboprobe were performed(FIG. 4 c, FIG. 11 c). ISH indicated that most of the embryos (<75%,n=10-16 per condition) treated with DAPT or FLI-06, -14, -15 and -20displayed expanded and denser clusters of ngn1 positive cells along thespinal cord, mostly in the motor neuron layer, compared to DMSO-treatedcontrol embryos (FIG. 4 c). Likewise, ngn1 positive cell clusters in thedeveloping brain of DAPT as well as FLI-06, -14, -15 and -20 treatedembryos were found differentially expanded compared to DMSO controls(FIG. 11 c). In contrast, the size of ngn1 positive clusters was reducedin FLI-16 and increased in FLI-19 treated embryos, although both did notinduce changes in qPCR analyses. Taken together, FLI-06, -14, -15, -19and -20 inhibited endogenous Notch signaling in vitro and in vivo,corroborating the NotchΔE-EGFP reporter data.

Small Molecule Hit Validation

From the above experiments, the dihydropyridine FLI-06 (1) emerged as aprominent hit compound with a novel mode of action. In order to confirmthe small molecule's structure, it was independently re-synthesizedde-novo, purified by re-crystallization, and stringently characterizedby X-ray crystallography. This substance was found to be equally activeas the initial screening hit. The clinically established Ca²⁺-Channelblockers nifepidine (ortho-NO₂ group—not encompassed by the chemicalformula of the present invention) and nimopidine (meta-NO₂ group—alsonot encompassed by the chemical formula of the present invention), twostructurally related 1,4-dihydropyridines, were tested alongside andfound completely inactive in our assay systems, showing that concomitantmodulation of Ca²⁺-signaling events is not causal for the observedphenotype (FIG. 5 a).

Preliminary SAR studies were then executed using a combination ofcompound acquisition and dedicated synthesis in order to gain insightinto the specificity of compound activity, and to ensure thatpotentially metabolically labile structural elements (NO₂ group,dihydropyridine ring) did not interfere with the phenotype observed.Activity was measured by determining enuc/nuc ratios in NotchΔE-EGFPcells (FIG. 5 a). Longer or lipophilic substituents at position R2according to formula I of the present invention were found to bepreferred for activity (Pent (3; FLI-27), cHex (1, 5; FLI-06, FLI-25)and cHept (4; FLI-28)), whilst the iso-pr group also showed the desiredeffect. Compound 4 (FLI-28) was twice as active as compound 1 (FLI-06).

The 4-NO₂ group is a preferred substituent and may be important for theobserved phenotype due to rendering its metabolic turnover unlikely.Conversion of the pendant keto group into an O-alkyl oxime againgenerated an entirely inactive substance (compound 6—not encompassed bythe chemical formula of the present invention). Surprisingly, FLI-06exerted a very unique effect in cells with a mode of action unrelated toknown targets of dihydropyridine scaffolds.

Further comparisons were carried out using several compounds of theprior art in order to assess whether the known compounds exhibited thedesired functional properties according to the present invention. Asshown in FIG. 5 b, the compounds HPI-1 from WO 2009/102864, compoundsC3, C4 and C5 from WO 2008/103470 and compound ST216093 from WO2008/070875 were tested in NotchΔE-EGFP cells. Compound E is provided asa positive control.

The specific structures of the compounds of the prior art are shown inFIG. 5 c. The compounds HPI-1, C3, C4 and C5 show no activity using theassay of the present invention. The compound ST216093 does however showthe desired activity. ST216093 has previously only been disclosed withrespect to the treatment of Alzheimer's disease, and is disclosed in WO2008/070875 as lacking any activity.

FLI-06 Leads to Disruption of the Golgi Apparatus Differently than BFA

At this stage, we intended to investigate the cellular activity of thedihydropyridine FLI-06 in more detail, namely the aberrant accumulationof NotchΔE-EGFP in intracellular membranes (FIG. 2 a). The aberrantglycosylation pattern of APP and Klotho as well as their reducedectodomain shedding (FIG. 3 a) suggested that FLI-06 interferednegatively with the secretory pathway. Strikingly, immunofluorescenceanalysis of HeLa cells with markers for ER (calnexin) and the Golgi(giantin) revealed that FLI-06 caused a complete disruption of the Golgiwhile the ER, at least when imaged at this resolution, seemed largelyunaffected (FIG. 5 d). Disruption of the Golgi after FLI-06 treatmentcould be caused by disassembly of the microtubules²⁸ or by interferingwith membrane trafficking in the early secretory pathway, similar toBrefeldin A (BFA²⁹) or Golgicide A (GCA³⁰).

In order to discriminate between these alternatives, cells wereincubated with FLI-06 or the microtubule-depolymerizing agent nocodazoleand analyzed by fluorescence microscopy (FIG. 12 a). While afternocodazole treatment the microtubule network was almost completelydisintegrated and mitotic spindles were absent in dividing cells, inboth treated as well as non-treated cells a fully intact microtubularcytoskeleton and mitotic spindles were observed. Actin polymerizationand distribution remained unaffected, which suggested that FLI-06 doesnot act on the cytoskeleton (FIG. 12 b). Similar to BFA, FLI-06 causeddispersal of the cis-Golgi marker GM130 and of the trans-Golgi network(TGN) marker TGN46 throughout the cytosol (FIG. 13). Both FLI-06 and BFAdid not affect endocytosis in a transferrin uptake assay. However, incontrast to BFA, FLI-06 did not cause tubulation of endosomes (FIG. 13).Another striking difference to BFA was observed when we assessed thedispersal of βCOP, a component of the COPI-derived coatomer complex³¹.While in BFA-treated cells the Golgi-like juxtanuclear βCOP staining wasalready lost after 10 min, it took 30-40 min in FLI-06-treated cells(FIG. 14 a). In contrast to BFA, treatment with FLI-06 did not lead tofusion of ER and Golgi (FIG. 14 b). Again in contrast to BFA, FLI-06 didnot cause a hyperglycosylation of VSVG-EGFP (FIG. 14 c), consistent withthe observation that Golgi-resident enzymes did not redistribute intothe ER. BFA acts by inhibiting several Arf-GEFs involved in varioustrafficking steps in the secretory pathway^(32,33). Golgicide A (GCA)inhibits only GBF1, the Arf-GEF acting on the early secretory pathway³⁰.To test whether FLI-06 acts on GBF1, HeLa cells were transfected withGBF1-GFP³⁴ and treated with BFA, GCA and FLI-06, respectively (FIG. 5e). In untreated cells GBF1-GFP displayed a cytosolic distribution,while in BFA- and GCA-treated cells it is recruited to the fragmentedGolgi. In contrast, in FLI-06 treated cells GBF1-GFP displayed thedistribution of control cells, suggesting that FLI-06 does not act onGBF1 and therefore different from BFA or GCA. We also noted that BFA andGCA, but not FLI-06 caused ER-stress as indicated by up-regulation ofthe ER-resident chaperone BIP and ATF6 luciferase assay (FIG. 15 c, d).Taken together, these data suggested that the dispersal of the Golgicaused by FLI-06 is not mediated by depolymerizing microtubuli anddiffers from a BFA-like mechanism.

FLI-06 Inhibits Cargo-Recruitment to ER Exit Sites

The differences between FLI-06 and BFA or GCA-treated cells prompted usto analyze the effect of FLI-06 on the first sorting/budding station inthe early secretory pathway, the ER exit sites (ERES). FLI-06 globallyaffects secretory transport of transmembrane (FIG. 3 a), secretory andGFP-anchored proteins (FIG. 15 a, b). Therefore, we selected thetemperature-sensitive VSVG-tsO45-mutant tagged with GFP (VSVG-EGFP³⁵) asgeneral reporter for secretory transport. VSVG-EGFP at 40° C. ismisfolded and accumulates in the ER. Upon shifting to the permissivetemperature (32° C.), a wave of VSVG-EGFP is exported from the ER andcan be followed in real-time^(35,36). VSVG-EGFP was transfected in HeLacells, incubated at 40° C. to accumulate it in the ER, and released toERES by shifting to 32° C. in the presence and absence of FLI-06 (FIG. 6a). To slow down export from ERES, cells were additionally incubatedwith nocodazole³⁷, which induces depolymerization of microtubuli butdoes not inhibit ER export. As described before³⁷, shifting untreatedcells to the permissive temperature resulted in the accumulation ofVSVG-EGFP in ERES, visualized by colocalization with the ERES markerSec31a (FIG. 6 a, a′, 30 min, arrowheads). After 85 min most ofVSVG-EGFP had left the ER and ERES and accumulated in post-EREScompartments devoid of Sec31a (FIG. 6 a′, 85 min, double arrows). Insharp contrast, in cells incubated with FLI-06, VSVG-EGFP remaineddiffusively distributed in the ER and almost no colocalization withSec31a was observed after 30 min. After 85 min some weak accumulation ofVSVG-EGFP in ERES was apparent (FIG. 6 a, a′, 85 min, arrowheads).Quantitation of the variance of pixel fluorescence intensity accordingto Dukhovny et al.³⁷ supported the observation that in control cellsVSVG-EGFP rapidly enters and leaves ERES, whereas in FLI-06 treatedcells the recruitment to ERES is strongly reduced (FIG. 6 b). Cellsshown in FIG. 6 were pretreated with FLI-06 at 40° C. for 30 min, toensure maximal inhibition. To get an initial idea on the velocity ofFLI-06 action, cells transfected with VSVG-EGFP were pretreated for 30or 10 min prior to the 32° C. chase, or FLI-06 was only added to thechase medium. In both cases ER export was inhibited to the same extendas with the 30 min preincubation, indicating that FLI-06 actsinstantaneously on ER export (FIG. 16). Interestingly, although noVSVG-EGFP and presumably no other cargo was recruited, Sec31a punctawere still observed after FLI-06 treatment (FIG. 6 a). Theimmunofluorescence data on VSVG-EGFP suggested that FLI-06 acted on apre-ERES step, such as cargo recruitment. Therefore, it should notaffect COPII budding from ERES. To test this hypothesis, awell-established in vitro budding assay using permeabilized cells, ratliver cytosol and an ATP regenerating system was employed. In this assayERGIC-53 and Sec22b served as markers for proteins incorporated intoCOPII vesicles and ribophorin I as a marker for an ER-residentprotein³⁸. This assay demonstrated that FLI-06 did not inhibit the COPIIbudding reaction when added directly to the assay (FIG. 6 c), whereasH89, a known inhibitor of Sar1-mediated ER-export³⁹, completely blockedformation of COPII vesicles. In contrast, when cells were pretreatedwith FLI-06 for 4 hours, the budding of COPII vesicles was slightlyinhibited in the absence of FLI-06 and strongly in the presence of 100μM FLI-06. As a control, all other cells were pretreated with FLI-25, aninactive derivative of FLI-06 (see FIG. 5 a). When added to the buddingreaction, neither 10 nor 100 μM of FLI-25 had an effect on the in vitroCOPII budding reaction. In summary, these data suggest that FLI-06inhibits ER-export at a pre-ERES step, potentially at the stage of cargorecruitment. They establish FLI-06 as the first small molecule probedecisively affecting this early step in the secretory pathway.

FLI-06 Converts Tubular ER to Sheets

We noted a morphological change of the ER upon FLI-06 incubation thatwas difficult to capture in fixed cells. Live-cell imaging was thereforeperformed in COS cells transfected with the ER marker prlss-KDEL-mRFP(supp. Ref 3) and FLI-06 was added for 120 min (FIG. 7 a). After 120 minalmost all cells had no detectable ER-tubules anymore and instead werefilled with large sheet-like structures. Sheet-formation started after5-10 min and had a t_(1/2) of about 14 min (FIG. 7 b). To test whetherthe morphological change is related to the inhibition of ER exit, nextour set of FLI-06 derivatives was tested. Only FLI-06, 3, 4 and 5, thosederivatives that were inhibiting ER exit, also elicited thetubule-to-sheets phenotype, strongly suggesting that both effects arerelated (FIG. 17). In addition, incubating cells with FLI-06 andcycloheximide demonstrated that the sheet formation is not the result ofcargo accumulation in the ER (FIG. 17). Because ER exit inhibitionprecedes sheet formation, the data suggested that sheet formation is notcausative for the ER exit block. Rather, the ER-sheets are indicativefor structural changes, maybe initiated within ERES, that inhibit ERexit.

FLI-06 Inhibits Secretion of Secreted Alkaline Phosphates

In order to confirm the inhibitory effect of FLI-06 on secretion wetransfected HeLa cells with a plasmid encoding a secreted alkalinephosphatase (SEAP). We collected the medium and measured SEAP secretionvia photometry and could confirm inhibition of secretion of SEAP uponFLI-06 incubation (FIG. 18 a).

FLI-06 Kills Cancerous T-Cells

To test whether FLI-06 would ultimately be useful to treat cancerousdiseases we incubated DND-41 cells having a Notch heterodimerizationdomain that leads to hyperactive Notch signaling (Weng et al, Science,2004) with FLI-06. 10 μM FLI-06 caused total cell death after 4 dayswhile 1 μM FLI-06 inhibited proliferation of DND-41 cells (FIG. 18 b).

Discussion of the Experimental Examples

The identification of small compounds specifically modulating abiological process constitutes a key step toward drug discovery. Here,we have developed and applied automated microscopy-based HCS to findnovel compounds affecting the Notch pathway. Notch signaling isimplicated in numerous developmental processes, differential decisionsand—not surprising for such an important pathway—is implicated in anumber of pathological conditions like neurodegeneration andT-ALL^(9,40). In the initial screen we intended to focus ontrafficking/processing aspects of Notch signaling. We used anEGFP-tagged reporter construct that was transcriptionally inactive. Thefluorescence of this Notch-based reporter was quantified in the nucleusand in a ring around the nucleus, to identify hit compounds. It shouldbe emphasized that they were extracted from the primary screeninglibrary, and were not further optimized yet. Despite this, FLI-06, -14,-15, -20, and less pronounced -19, did not show acute toxicity on thetime scale of our experiments, and clearly reduced endogenous Notchsignaling, as shown by reduction of CSL-dependent luciferase-activity inC2C12 cells and by causing somite malformation and neurogenesisphenotypes in vivo in zebrafish.

We found that FLI-06 generally blocked secretion and that the GSIsFLI-14 and -19 as well as FLI-15 and -20 inhibited Notch and APPprocessing, indicating they are not specific for Notch. Nevertheless,the dominant phenotype of all five compounds observed in vivo was aNotch phenotype, suggesting that future structure-function analysestogether with time and dose-optimizations should enable the developmentof probes interfering more specifically with Notch signaling.

The active probes identified acted on different steps in trafficking andprocessing of the reporter (schematized in FIG. 18). Four compoundscaused accumulation of the Notch-reporter at the PM, suggesting thatγ-secretase itself is inhibited or trafficking/interaction ofγ-secretase with the Notch-reporter. Indeed, two of the four compounds(FLI-15 and FLI-20) turned out to be novel bona-fide GSIs, but whetherthey directly inhibit γ-secretase or act indirectly or allostericallyremains to be addressed. While GSIs are probably not primary drug leadsfor Alzheimer disease⁴¹, they show promising therapeutic potential forT-ALL⁴². In addition, GSIs proved to be instrumental forcharacterization of the different subunits of γ-secretase, for exampleallosteric interaction sites and substrate binding sites¹³.Interestingly, FLI-14 and -19 did not inhibit γ-secretase at theconcentrations used in the in vitro assay, despite the fact that theirapplication resulted in accumulation of the reporter at the PM and theirclear effect on endogenous Notch signaling. Potentially, thesesubstances could affect the recruitment of substrate to γ-secretase, itstargeting to the PM and/or endosomes, or the targeting of substrate orenzyme to detergent-resistent membrane domains where active γ-secretaseresides⁴³.

Because of its striking phenotype, namely the accumulation ofNotchΔE-EGFP in intracellular membranes, the dihydropyridine FLI-06 wasstudied in more detail. Related 1,4-dihydropyridines such as nifedipineare widely applied as drugs in humans to treat hypertension and aregenerally recognized as Ca²⁺-channel modulators with antagonistic oragonistic activity⁴⁴, but are inactive in our settings. Otherphysiological activities for dihydropyridines have been investigated,most notably anti-atherosclerotic, hepatoprotective, anti-mutagenic, andanti-diabetic properties⁴⁵. Some of these activities could be related tothe antagonistic activity some dihydropyridines show on themineralocorticoid receptor^(46,47). While the extent of these effects isknown to strongly vary with small changes in molecular structure ofdihydropyridines⁴⁸, specific activity on intracellular trafficking of adihydropyridine scaffold was completely unprecedented. Similar to BFAand GCA or probes like the PKA inhibitor H89, treatment of cells withFLI-06 resulted in disruption of the Golgi apparatus. However, ourexperimental data stringently suggested that FLI-06 acted via adifferent mechanism. FLI-06 did not affect the recruitment of GBF1 tothe Golgi, the target of BFA and GCA. In contrast to BFA, the Golgi didnot fuse with the ER in the presence of FLI-06, and the kinetics ofβ-COP dissociation and Golgi dispersal differed between FLI-06 and BFA.Unlike H89, FLI-06 did not directly inhibit COPII budding in vitro.Further studies with VSVG-EGFP suggested that FLI-06 acted on a veryearly step in recruitment of cargo to ERES.

Mechanistically, the formation of ERES and initiation of cargorecruitment starts with the recruitment of Sar1 by Sec12. Sar1 in turnrecruits the cargo receptors Sec23/24. Finally, Sec13/31 are recruitedand the fission of a COPII vesicle is initiated (for review see⁴⁹). Inthe in vitro COPII budding assay pre-incubation of the cells wasrequired to see a block in vesicle formation. This result suggested thatFLI-06 does not affect the essential proteins provided by the addedcytosol in the budding reaction. At the present stage we hencehypothesize that FLI-06 acts on the level of Sec12 or other currentlyunknown recruitment factors—or on the membrane structuring eventsnecessary to initiate an ERES. Although no VSVG-EGFP, no NotchΔE-EGFPand presumably no other cargo accumulates at ERES, there are still Sec31labeled ERES, suggesting that cargo recruitment is not essential forrecruiting COPII components to ERES. Strikingly, inhibition of ER exitwas followed by a complete tubule-to-sheet transition of the ER.Morphological changes in ER structure can be caused, among others, bydisrupting ER-microtubule connections (Klopfenstein, 1998) or byinterfering with structural proteins in the ER (Shibata, 2009; Voeltz,2006). Depolymerizing microtubules did not affect secretion (Rogalski,1984; Cole, 1996). Sheet formation alone, induced by microtubuledepolymerization or interfering with ER-microtubule interactingproteins, does not inhibit ER exit (FIG. 7C) and clearly occurs afterblocking secretion. It is not just an indicator of accumulating cargo,and occurs only with FLI-06 and its derivatives that block secretion,strongly suggesting that the two effects are related. ERES are highlycurved membrane regions, and we speculate that FLI-06 causes somecurvature changes in ERES that inhibit cargo recruitment. Curvaturechanges spread then all over the ER, leading to the observed sheetformation. Interestingly, while this study was in revision, the smallmolecule dispergo was discovered (Lu, 2013). Dispergo has the oppositeeffect since it induces ER tubulation, but like FLI-06 it seems toinhibit recruitment of cargo to ERES. This might indicate that the“correct” membrane curvature at ERES is essential, and shifts in eitherdirection disrupt ER exit.

The activity of the small molecule probe FLI-06 (1) was furthervalidated by resynthesis and focused structure variations (compounds2-5). These initial experiments on structure-function relationships ofFLI-06 showed that larger or bulky alkyl residues increased activity. Atthe present stage, a p-NO₂ group appears to be important but notessential for compound activity. Metabolic modification (oxidation orreduction) of the scaffold seems unlikely, given the rather narrowactivity window and fast onset of activity. In addition, the respectivederivatives were inactive. Further compounds according to the chemicalformulae described herein have been synthesized (see below forinformation on chemical synthesis) and experimental analysis is ongoing.

Taken together, FLI-06 is a unique chemical for the treatment ofsecretion-dependent disease. To our knowledge, FLI-06 is the onlycompound that acts this early in the secretory pathway, at pre-ERESsteps. In addition, and as an additional benefit, FLI-06 does not causesignificant ER-stress, in contrast to BFA or GCA, thereby indicatingreduced side effects after medical administration.

Methods and Materials Applied in the Examples of the Present Invention

cDNAs and Antibodies

Antibodies and cDNAs used in this work are listed in a table below.

Maintaining of Cell Lines and Generation of Stably Expressing Cell Lines

Cells were maintained in Dulbecco's modified Eagle Medium+GlutaMax(Invitrogen) supplemented with 10% FBS. For stable lines, HeLa Kyoto andU2OS cells were transfected with NotchΔE-EGFP with Lipofectamine 2000(Invitrogen), sorted via FACS and selected with 100 μg/ml Hygromycin B.Single cell clones were picked and selected based on moderate andhomogenous NICD-EGFP nuclear staining. One clone was then selected forfurther use.

ChemBioNet Compound Screen

The compound screen was performed at the Leibniz-Institut für MolekularePharmakologie (FMP) in Berlin as a single screen, measuring the enuc/nucratio of the GFP signal. The compounds were applied on 51 screeningplates at 10 iM for 24 h and processed for image acquisition andanalysis. The Z′ for the individual plates ranged between 0.4 and 0.8(=0.53±0.14), indicating excellent assay conditions with only two platesfalling below that range. Activity was assessed by z-score normalizationand samples with less than 150 cells were dismissed from furtheranalysis. For hit validation compounds were ordered from ChemDiv or wereobtained by chemical synthesis. For further details see methods below.

EC₅₀ Determinations

EC₅₀ values of the test compounds were calculated from serial dilutionseries ranging from 200-0.1 μM. Cells were seeded in 96-well plates at adensity of 5000 cells/well in 100 μl medium. The next day, 100 μl mediumcontaining the respective test compounds was added. Cells were incubatedfor 16 h, fixed and processed for automated microscopy. For the putativegamma-secretase inhibitors the enuc intensity was divided by the DAPTcontrol and for the trafficking inhibitors normalized percentageinhibition against DAPT/DMSO controls of log 2 transformed nuc/enucratios were calculated. Relative activity values were read into “R”(http://www.r-project.org/) and EC₅₀ estimates were calculated usingfour-parameter log-logistic fit with the package “drc”⁵¹.

Drug Treatments

If not stated otherwise, all drugs were purchased from Sigma Aldrich.Drugs were used at the following concentrations. BFA, 1 μg/ml; GolgicideA (Calbiochem), 10 μM; nocodazole, 1.5 μg/ml; H89, 25 μM; tunicamycin,10 μg/ml; DAPT (Alexis Biochemicals), 1-2 μM

In Vitro γ-Secretase Assay

For assaying AICD formation, membranes of HEK293 cells stably expressingAPP with the Swedish mutation were isolated and incubated for 4 h at 37°C. according to Sastre et al.⁵². For assaying NICD formation, membranesfrom HeLa NotchΔE-EGFP cells were mixed with membranes from HeLaNotchΔE-EGFP cells that were pre-treated with 10 μM DAPT overnight toenrich substrate. After incubation samples were loaded onto either 8%SDS-PA gels (NICD) or 10-20% Tris-Tricine gels (AICD), blotted andprobed with cleaved-Notch antibody or antibody 6687 against APPC-terminus⁵³. Chemoluminescence was quantified on a LAS-4000 (Fuji) withMultiGauge software.

Detection of APP, Klotho and their Cleavage Products

APP, APPs, APP_(CTF) and Aβ detection was as described before⁵⁴ usingantibodies 22C11 for APP and APPs, 6687 for APP_(CTF) and 3552 and 2D8for Aβ. Klotho was detected as described in Bloch et al.²⁰.Chemoluminescence was quantified on a LAS-4000 (Fuji) with MultiGaugesoftware.

Luciferase Assay

Endogenous Notch signaling in C2C12 was determined by a luciferase assayusing a 12xCSL-luciferase reporter and transfected Delta as describedbefore¹⁵.

VSVG-Assay

HeLa cells plated on cover slips were transiently transfected withtemperature sensitive VSVG-tsO45-mutant carrying an EGFP-tag (plasmidVSVG3-GFP³⁵). After 24 h cells were transferred to 40° C. for 24 h toaccumulate VSVG-EGFP in the ER. Before the chase, nocodazole (1 μg/ml)and DMSO or BFA (1 μg/ml) or FLI-06 (10 μM) were added and cells wereincubated on ice for 30 minutes to depolymerize microtubules. For thechase, cells were transferred to a waterbath with 32° C., fixed afterindicated time points and stained with an antibody against Sec31 todetect localization of ERES. During 0° and 32° C. incubations 10 mMHEPES was added.

Transferrin-Uptake

For the transferrin uptake assay cells were starved in serum free mediumfor 1 h at 37° C. Cells were then transferred on ice and medium wasexchanged to serum free medium supplemented with 25 μg/mlAlexaFluor555-Transferrin conjugate (Molecular Probes) and testcompounds. After 15 min on ice cells were incubated with pre-warmedserum-supplemented medium containing test compounds and incubated at 37°C.

Fluorescence Microscopy

Immunofluorescence stainings were made using standard procedures⁵⁵.Imaging was performed on a Zeiss Axiovert200 or an Axio Imager, using63× 1.4NA objectives and Zeiss Axiovision software. For live-imagingcells were plated on Lab-Tek chambered coverglass (Thermo-Fisher).Images were assembled and processed using Adobe Photoshop. Fordisplaying weakly stained ER-tubules/sheets non-linear changes ingamma-settings were used.

Compound Identity

Identity and purity of purchased compounds was verified by thin layerchromatography and mass spectrometry. Chemically synthesized compoundswere spectroscopically characterized.

In Vitro Budding Assay

COPII budding in vitro was essentially performed as described in Kim etal.³⁸.

Zebrafish

Details of zebrafish experiments can be found below.

Statistical Analysis

Means of numerical data were compared using Student's t-test. Adifference in means was considered statistically significant (*) withp<0.05 or p<0.01 as indicated. Error bars depict the standard error(SEM) or standard deviation (SD) as indicated. The number of independentreplicates is also indicated in the figure legends.

Antibodies

Antibody supplier, order number or (target/markerfor) species,poly/monoclonal reference Sec31a (ERES) mouse, mono BD, #612350 Klothogoat, poly R&D Systems, AF1819 Klotho rat, mono KM2119 (Supp. Ref 1)actin rabbit, poly Abcam, ab8227 NICD (V1744) rabbit, poly CellSignaling, #2421 6687 (APP rabbit, poly gift from C. Haass, C-terminus)(Supp. Ref 2) BIP/GRP-78 (ER) goat, poly Santa Cruz, sc-1051 β-COP(Golgi) rabbit, poly ThermoFisher, PA1-061 Giantin (Golgi) mouse, monoEnzo, ALX-804-600 Calnexin (ER) mouse, mono Chemikon/MilliPore, MAB3126FLAG mouse, mono Sigma F3165 ERGIC53 (ERGIC) rabbit, poly Schekman Lab2925/2926 LAMP1 (lysosom) rabbit, poly abcam, ab19294 GM130 (cis-Golgi)mouse, mono BD, 610822 GFP rabbit, poly Invitrogen A11122 a-tubulinmouse, mono Sigma T9026, DM1a TGN38 (TGN) rabbit, poly Santa Cruz,sc-33783 3552 (Aβ) rabbit, poly gift from C. Haass 2D8 (Aβ) rat, monogift from C. Haass 22C11 (APP, APPs) mouse, mono Millipore Plasmid(marker for) provided by prlss-KDEL-mRFP3 (ER) Erik Snapp, AlbertEinstein College of Medicine New York, NY 10461, USA [Snapp, 2006 #3402]p5xATF6-GL34 (ER stress Addgene #11976 indicator) pGL4, 74 (Renillaexpression Promega control)

ChemBioNet Compound Screen (See Also Table 2)

For the compound screen 3000 cells were pre-seeded in 384-well plates(Corning, Corning, N.Y.) in DMEM+10% FBS with a multi dispenser. Thenext day, compounds were added from a 1 mM stock library at 0.5 il to afinal concentration of 10 iM per well. Controls were added with amultichannel pipette at 21M for DAPT and 1% final DMSO in 50 il.Pipetting was performed with a Caliper robot. The plate layout included16 wells for each control. Plates were incubated for 24 h at 37° C., 95%relative humidity, 5% CO2. After incubation cell culture medium wasaspirated and replaced with 25 il 4% formalin for 20 min. After fixationcells were washed once with PBS and nuclei were stained with 51M Hoechstfor 20 min. After another washing step cells were covered with 25 ilPBS. The screened library was described (ref 17).

HCS Image Acquisition and Data Analysis

Images were acquired on an ArrayScan VTi automated microscope(ThermoFisher) and numerical data were extracted with the CompartmentalAnalysis BioApplication of the bundled software suite. Compoundscreening raw data were collected at the FMP and data transformation wasperformed according to standard procedures (ref 17 and supp. Refs 6-7).

Identity and Purity of Commercially Acquired Samples

The purity of the compounds was tested by using thin layerchromatography (TLC) and mass spectroscopy (MS), which additionallyallowed checking the identity. TCL analysis was performed with silicagel 60 F254 aluminum sheets (Merck). Chloroform/methanol mixtures wereused as eluent. By UV illumination and iodine staining no impuritieswere detected. MS was performed on a TRIO 2000 (Fisons) spectrometer inEI ionization mode at 70 eV.

No. OrderID and Vendor MW Formula m/Z found Purity MS FLI-06 1630-0135438.53 C25H30N2O5 438 OK ChemDiv FLI-14 C329-0322 512.81 C30H32N4O4 512OK ChemDiv FLI-15 C548-2756 405.49 C17H19N5O3S2 405 OK ChemDiv* FLI-194464-0971 565.70 C32H43N3O6 588 (M + Na+) OK ChemDiv FLI-20 STK164160383.51 C21H25N3O2S 438 Impurity with Vitas-M M = 438.6 FLI-24BAS00087237 398.45 C22H26N2O5 398 OK (3) Asinex FLI-25 1630-1646 393.52C25H31NO3 393 OK (11) ChemDiv FLI-27 OSSL_264545 426.51 C24H30N2O5 426OK (4) Princeton Biomol. Res. FLI-28 OSSK_158427 452.23 C26H32N2O5 452OK (5) Princeton Biomol. Res. *The ChemDiv entry containsC15H19CIN4O5S3, MW = 466.02; the correct structure was determined byNMR.

Compound Synthesis

Unless otherwise noted, all commercially available compounds were usedas provided without further purifications. Reactions were monitored byTLC on 0.2 mm Merck silica plates (60, HF254). 1H and 13C NMR spectrawere recorded on Bruker AVANCE 250 or 400 spectrometers, chemical shiftsare given relative to residual solvent signals. Melting points wererecorded in open capillaries and are uncorrected. Mass spectra wereobtained on a TRIO 200 from Fison or on a FINNAGEN MAT DDQ 710.Anhydrous solvents were obtained following general laboratory procedures(supp. Ref 8). Beta-ketoesters were obtained from2,2,6-trimethyl-4H-1,3-dioxin-4-one and the respective alcohol followingpublished procedures (Supp. Ref 9) and distilled before use. Ammoniumacetate was purified and dried by sublimation.

Representative Procedure for Dihydropyridine Synthesis.

Loosely following the precedent of Gestwicki (supp. Ref 10) dimedon D(7.1 mmol, 1.0 g), the respective β-ketoester A (4 mmol) and ytterbiumtriflate (0.32 mmol, 8 mol %, 0.2 g) were dissolved in anhydrousacetonitrile (25 mL) and stirred under nitrogen for 10 min. A cold (4°C.) solution of anhydrous NH4OAc B (5.6 mmol, 0.3 g) in methanol (10 mL)was introduced. After 10 min the corresponding aldehyde C (4 mmol,dissolved in 10 ml acetonitrile) was added dropwise. The yellowishmixture was stirred at room temperature overnight, then poured intowater (100 mL) and stirred for one hour. The precipitate formed waseither filtered of by suction or extracted with ethyl acetate.

The remaining material was dissolved in ethyl acetate/hexanes andfiltered over a short column of silica. The solvent was removed, theresidue recrystallized form acetonitrile and dried in vacuo.Transformations were generally cleaner when the enamineoester E wasindividually formed (Supp. Ref 11). As side products, symmetrical doubleadducts of dimedone and aldehyde were observed in varying amounts (seesupporting scheme 1) and individually isolated for comparative testing(see below).

Ethyl4-(4′-nitrophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate

Light yellow crystals; yield: 61%; m.p. 188° C.; 1H NMR (250 MHz,CDCl3): ä 8.09 (d, J=8.7 Hz, 2H), 7.49 (d, J=8, 7 Hz, 2H), 6.93 (br s,1H), 5.16 (s, 1H), 4.09 (q, J=7.0 Hz, 2H), 2.41 (s, 3H), 2.28-2.10 (m,4H), 1.25 (t, J=7, 2 Hz, 3H), 1.09 (s, 3H), 0.91 (s, 3H); 13C NMR (62.5MHz, CDCl3): ä 195.1, 166.7, 154.2, 146.2, 144.2, 128.9, 123.3, 111.3,105.1, 60.1, 50.5, 41.2, 37.2, 32.7, 29.3, 27.1, 19.5, 14.2; MS (EI):m/z (%) 384 (M+) (66), 355 (21), 262 (100), 234 (81), 178 (29), 150(17), 83 (9).

Cyclohexyl4-(4′-nitrophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate(1, “FLI-06”)

Light yellow crystals; yield: 51%; m.p. 196° C.; 1H NMR (250 MHz,DMSO-d6): ä 8.09 (d, J=8.8 Hz, 2H), 7.51 (d, J=8.8 Hz, 2H), 5.94 (s,1H); 5.16 (s, 1H), 4.69-4.69 (m, 1H), 2.42 (s, 3H), 2.33-2.16 (m, 4H),1.80-1.21 (m, 10H), 1.09 (s, 3H), 0.90 (s, 3H);

13C NMR (62.5 MHz, CDCl3): ä 195.2, 166.2, 154.3, 148.3, 146.2, 144.0,129.9, 123.4, 111.2, 105.3, 72.5, 50.6, 41.2, 37.3, 32.7, 31.8, 31.5,29.3, 27.1, 25.3, 23.8, 23.6, 19.5; MS (EI): m/z (%)=438 [M+] (67); IR(ATR, [cm-1]): 3198 (m), 3088 (w), 2937 (m), 1672 (s), 1600 (s), 1482(s), 1468 (m), 1340 (vs), 1433 (vs), 1171 (m), 1107 (s); analysis calcd.for C25H30N2O5: C, 68.47; H, 6.90; N, 6.39. found 68.7, 7.3, 6.5.

Cyclohexyl4-(4′-cyanophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate

Colourless crystals; yield: 88%; m.p. 235° C.; 1H NMR (250 MHz, CDCl3):ä 7.52 (d, J=8.3 Hz, 2H), 7.45 (d, J=8.3 Hz, 2H), 5.75 (s, 1H), 5.10 (s,1H), 4.99-5.12 (m, 1H), 2.42 (s, 3H), 2.32-2.27 (m, 4H), 1.81-1.25 (m,10H), 1.09 (s, 3H), 0.90 (s, 3H); 13C NMR (101 MHz, CDCl3,): ä 195.2,166.2, 152.2, 147.9, 143.9, 131.8, 128.9, 111.4, 109.6, 105.4, 72.4,50.6, 41.2, 37.3, 32.7, 31.8, 31.4, 29.3, 27.1, 25.3, 23.7, 23.6, 19.6;MS (DEI): m/z (%)=416 (M+) (24), 334 (100), 317 (20), 278 (37), 260 (9);fluorescence (CH2Cl2): └max.: 432 nm; fluorescence excitation (CH2Cl2):└max.: 371 nm.

Cyclohexyl2,7,7-trimethyl-5-oxo-4-(pyridin-4-yl)-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate

Light yellow crystals; Yield: 33%; m.p. 238° C.; 1H NMR (250 MHz,CDCl3): ä 8.44 (d, J=6.0 Hz, 2H), 7.28 (2H, J=6.1 Hz, 2H), 6.32 (s; 1H),5.07 (s, 1H), 4.60-4.69 (m, 1H), 2.42 (s, 3H), 2.32-2.17 (m, 4H),1.83-1.21 (m, 10H), 1.08 (s, 3H), 0.90 (m, 3H); 13C NMR (101 MHz,CDCl3,): ä 195.2, 166.2, 155.4, 149.2, 148.5, 144.4, 123.4, 110.9,104.8, 72.4, 50.6, 41.1, 36.6, 32.7, 31.8, 31.4, 29.3, 27.0, 25.3, 23.7,23.6, 19.4. MS (Micro-ESI): m/z (%)=417 (M+Na)+395 (M+H)+(54); HRMS:calcd. for [M+H]+C24H31N2O3=395.2334. found: 395.2329.

Cyclohexyl4-(4′-thioamidophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate

Nitrile 7 (1.2 mmol, 0.5 g) was dissolved in 25 ml DMSO and ammoniumsulphide solution (6 ml, 48%) was added with stirring. The pale greenmixture was stirred for one hour. Ice cold water was added (100 mL), andstirring was continued for 30 min. The crude product was recovered byfiltration and purified by recrystallization from ethanol/water (2:1).Bright yellow needles; yield: 92%; m.p. 231-235° C.; 1H NMR (250 MHz,DMSO-d6): ä 9.69 (s, 1H), 9.32 (s, 1H), 9.07 (s, 1H), 7.71 (d, J=8.3 Hz;2H), 7.17 (d, J=8.3 Hz, 2H), 4.87 (s, 1H), 4.58 (br s; 1H), 2.44-2.12(m, 7H), 1.92-1.24 (m, 10H), 0.99 (s, 3H), 0.82 (s, 3H); 13C NMR (101MHz, DMSO-d6): ä 200.4, 194.7, 166.5, 151.2, 150.2, 145.8, 137.5, 127.4,127.4, 109.9, 103.8, 71.4, 50.6; 36.5, 32.6, 31.7, 31.4, 29.5, 26.9,25.4, 23.6; MS (EI): m/z (%)=453 (M+) (18), 369 (9), 316 (82), 234(100), 190 (11), 83 (15).

Cyclohexyl2,7,7-trimethyl-4-(4′-nitrophenyl)-5-oxo-5,6,7,8-tetrahydroquinoline-3-carboxylate(7)

Dihydropyridine 1 (0.436 mg, 1 mmol) was dissolved in dichloromethane(50 mL) and MnO2 (excess, approx. 1 g) was added. The mixture wasstirred until the starting material was completely consumed (TLC,hexanes/EtOAc 1:1). The inorganic material was filtered off, solvent wasevaporated and the crude compound purified by column chromatography(SiO2, solvent hexanes/EtOAc 1:1). Off-white solid, yield 90%; 1H NMR(250 MHz, DMSO-d6): ä 8.26 (d, J=8.5 Hz, 2H), 7.32 (d, J=8.7 Hz, 2H),4.66-4.69 (m, 1H), 3.11 (s, 2H), 2.63 (s, 3H), 2.47 (s, 2H), 1.58-1.20(m, 10H), 1.12 (s, 6H); 13C NMR (101 MHz, DMSO-d6): ä 197.3, 166.1,163.1, 158.1, 147.3, 145.5, 145.2, 129.7, 129.2, 123.1, 122.6, 74.1,53.0, 46.9, 32.6, 20.9, 28.1, 26.8, 25.0; MS (EI): m/z (%)=436 (M+)(37), 353 (32), 316 (100), 309 (7), 234 (91).

Cyclohexyl-4-(4′-aminophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydro-quinoline-3-carboxylate

A schlenk-flask was purged with nitrogen and a solution of nitroarene 1(0.408 mg, 1.00 mmol) in MeOH (30 ml, anhydrous, degassed) wasintroduced, followed by Pd/C (5% on charcoal (10 mg). Hydrogen gas wasintroduced (1 bar) and conversion followed by TLC (EtOAc/PE, 1:1). Afterturnover was complete (2 h) the mixture was filtered and the solvent wasevaporated. The crude product was purified by radial chromatography(Chromatotron®) under N2-Atmosphere (CH2Cl2/MeOH 99:1), then dissolvedin EtOH (1 mL), triturated with cyclohexane and dried in vacuo.Off-white powder sensitive to air, must be stored below ambienttemperature; yield 95%; 1H NMR (250 MHz, DMSO-d6): ä 7.01 (d, J=8.3 Hz,2H), 6.54 (d, J=8.3 Hz, 2H), 5.79 (br s, 1H), 4.93 (s, 1H), 4.69-4.66(m, 1H), 2.36 (s, 3H), 2.27-2.17 (m, 4H), 1.62-1.24 (m, 10H), 1.07 (s,3H), 0.94 (s, 3H); 13C NMR (101 MHz, CDCl3): ä=200.4, 194.7, 166.5,151.2, 150.2, 145.8, 137.5, 127.4, 127.4, 109.9, 103.8, 71.4, 50.6,36.5, 32.6, 31.7, 31.4, 29.5, 26.9, 25.4, 23.6; MS (EI): m/z (%)=408(M+) (8), 406 (6), 390 (8), 392 (5), 318 (5), 316 (100), 234 (92), 216(25), 177 (16), 93 (97).

E-2-(((3′-Cyclohexyloxycarbonyl-4″-nitrophenyl-2′,7′,7′-trimethyl-1′,4′,5′,6′,7′,8′-hexahydroquinoline-5′-ylidene)amino)oxy)aceticacid (6)

A solution of O-(Carboxymethyloxmethyl)hydroxylamine hemihydrochloride(2.06 mmol, 225 mg, Aldrich) in water (5 mL) was adjusted to pH 5 withsolid sodium carbonate. This solution was evaporated to dryness in vacuoand the residue was suspended in methanol (6 mL).

To a solution of ketone 1 (0.5 mmol, 219 mg) in acetonitrile (8 mL),phosphorus oxychloride (5 mmol, 500 iL) was added under nitrogen at 30°C. and brought to reflux for 3 h. The orange reaction mixture wasevaporated at 50° C. i. V. (6 mbar) and dried until odorless (POCl3).The dark residue was dissolved in acetonitrile (6 mL) and freshlyprepared methanolic O-(carboxymethyloxmethyl)hydroxylamine solution (6mL, see above) was added at once. The mixture was heated to reflux for 5min before all the solvents were evaporated. The residue was taken upwith 20 mL of ethyl acetate, washed with water (2×10 mL), brine (1×10mL), dried with MgSO4, and evaporated to dryness. The residue wasdissolved in 3 mL of methyl tert-butyl ether (MTBE) and stored at 5° C.for 4 days. The crystalline product was retrieved by filtration anddried in vacuo to yield 148 mg of oxime 10. On concentration a secondcrop of 10 could be obtained by recrystallization from MTBE/n-hexane.

Amber colored crystals; yield: 58%; m.p. 129-133° C.; 1H NMR (250 MHz,CDCl3): ä 8.02 (d, J=8.5 Hz, 2H), 7.43 (d, J=8.5 Hz, 2H), 5.72 (s, 1H);5.06 (s, 1H), 4.70-4.60 (m, 1H), 2.23 (dd, J=4, 142.5 Hz, 2H), 2.28 (dd,J=5.2, 16.7 Hz, 2H), 2.36 (s, 3H), 2.33-2.16 (m, 4H), 1.80-1.21 (m,10H), 1.06 (s, 3H), 0.85 (s, 3H); 13C NMR (CDCl3, 62.5 MHz): ä 173.8,166.2, 155.9, 154.9, 146.0, 144.7, 137.2, 129.3, 122.8, 105.7, 102.6,72.9, 70.1, 40.7, 38.4, 36.2, 31.9, 31.7, 30.45, 29.5, 27.1, 25.4, 23.9,23.8, 20.1; ESI HRMS: calcd for (M++H+): C27H34N3O7: 512.2390. found:512.2390; analysis calcd. for C27H33N3O7×MTBE: C, 64.0; H, 7.6; N, 7.0.found C, 63.5; H, 8.0; N, 6.9.

Typical side products of Hantzsch-type dihydropyridine syntheses.

2,2′-((4-Nitrophenyl)methylene)bis(3-hydroxy-5,5-dimethylcyclohex-2-enone)

Obtained from the representative procedure for dihydropyridinesynthesis. After completion the crude mixture was recrystallized fromEtOH/H2O. The first crop was recrystallized again to afford the puretitle compound. Off-white crystals; yield: 30%; 1H NMR (250 MHz, CDCl3):ä=11.80 (s, 1H), 11.22 (br s, 1H) 8.15 (d, J=8.8 Hz, 2H), 7.26 (d, J=8.8Hz, 2H), 5.55 (s, 1H), 2.54-2.29 (m, 8H), 1.24 (s, 6H), 1.12 (s, 6H);13C NMR (62.5 MHz, CDCl3): ä=190.9, 189.6, 146.5, 146.1, 127.6, 123.5,114.9, 46.9, 46.4, 33.2, 31.5, 29.5, 27.4; MS (ESI): m/z (%)=436.2(M+Na)+; analysis calcd. for C23H27NO6: C, 66.8; H, 6.6; N, 3.4. foundC, 66.6; H, 6.4; N, 3.2.

2,2′-((3-Nitrophenyl)methylene)bis(3-hydroxy-5,5-dimethylcyclohex-2-enone)

Obtained from the representative procedure for dihydropyridinesynthesis. After completion the crude mixture was recrystallized fromEtOH/H2O. The first crop was recrystallized again to provide the puretitle compound. Light yellow crystals; yield: 61%; m.p. 202° C.; 1H NMR(250 MHz, CDCl3): ä 11.8 (s, 1H), 11.3 (br s, 1H), 8.06 (m, 2H), 7.45(m, 2H), 5.54 (s, 1H), 2.54-2.29 (m, 8H), 1.28 (s, 6H), 1.12 (s, 6H);13C NMR (61.5 MHz, CDCl3): ä 191.0, 189.6, 148.4, 140.7, 132.8, 129.1,122.2, 121.0, 114.8, 46.0, 46.4, 32.9, 31.4, 29.7, 27.3.

Crystal Structure Analysis Data of FLI-06

Intensity data were collected on a Nonius Kappa CCD diffractometer usinggraphite-monochromated Mo—K<radiation. Data were corrected for Lorentzand polarization effects but not for absorption effects (COLLECT, DataCollection Software; Nonius B.V., The Netherlands (1998) (Supp. Ref 12).The structures were solved by direct methods (SHELXS) (supp. Ref 13) andrefined by full-matrix least squares techniques against Fo (supp. Ref13) (SHELXL-97). All hydrogen atoms were located by difference Fouriersynthesis and refined isotropically. All non-hydrogen atoms were refinedanisotropically. Crystallographic data as well as structure solution andrefinement details are summarized in table 3. XP (SIEMENS AnalyticalX-ray Instruments, Inc.) was used for structure representations. Seealso FIG. 19.

Zebrafish:

Embryos were obtained from natural spawning of wild-type TüAB strainadults, raised and staged according to supp. Ref 14. DAPT and compoundswere applied at 50 iM in E3 embryo medium to zebrafish embryos withchorions torn but not completely removed from sphere stage until thestage of analysis, according to ref 23. Control embryos were mocktreated with the same concentration of DMSO dissolved in E3 embryomedium. All embryos were incubated in a 24-well plate (10-15embryos/well; 2 ml final volume) at 28° C. until analysis and then fixedin ice-cold buffered 4% paraformaldehyde overnight. Whole-mount in situhybridizations (ISH) were performed essentially as described (supp. Ref16). Digoxigenin-labeled antisense riboprobes were generated fromlinearized vectors as described (ref 26). For qRT-PCR analysis total RNAwas isolated from five zebrafish embryos showing similar phenotype usingthe RNeasy Mini Kit (Qiagen). In order to discard unwanted or toxiceffects, for qPCR those compound-treated embryos were selected thatdisplayed mild somite defects (see FIG. 11). Subsequent cDNA synthesiswas performed using the SuperScript III RT kit (Invitrogen), randomhexamer primers (Promega) and 500 ng total RNA as template. Quantitativereal-time RT-PCR analysis of cDNA was carried out using the SYBR greenERqPCR super mix for iCycler (Invitrogen) in an iCycler device (96-wellformat; Biorad). All samples were measured as triplicates and normalizedto the corresponding amounts of ef1a cDNA measured within the sameplate. Relative expression levels where calculated using the 2-ÄÄCTmethod (supp. Ref 18). For imaging embryos were washed twice in E3embryo medium and treated with a 0.016% tricaine (MS-222, Sigma)solution in E3 embryo medium. Then embryos were embedded in 3%methylcellulose for imaging. Live embryos (20 to 24 hpf) were scored formorphologic defects using an epifluorescence Stereo Discovery V8microscope (Carl Zeiss). Images were generated using the AxioVisionsoftware (Zeiss). Images of ISH were taken in embryos mounted in 70%glycerol/PBST.

TABLE 2 Small molecule screening data. Category Parameter DescriptionAssay Type of assay Cell based, image based Target Notch-traffickingPrimary measurement EGFP fluorescence Key reagents DMSO, DAPT Assayprotocol Measurement of nuc/enuc fluorescence intensity ratio of aGFP-tagged Notch reporter Additional comments Library Library sizeChemBioNet Library (Lisurek, 2010). 16,671 compounds. Librarycomposition Chemical Diversity, Bioactivity enriched. Source ChemDiv(San Diego) through Leibniz-Institute for molecular pharmacology,Berlin, Additional comments Screen Format 384 well microtiter platesConcentration(s) tested 10 μM Plate controls 3000 cells were preseededin 384 well plates. The next day compounds were added from a 1 mM Stockto yield 10 μM final concentration in 50 μl total volume. Plates wereincubated for 24 h at 37° C., 95% RH, 5% CO2 and processed as follows.Medium was aspirated, cells were fixed in 4% formalin for 20 min,stained with Hoechst 33342, rinsed and covered with PBS.Reagent/compound dispensing system Caliper (PerkinElmer) Detectioninstrument and software ArrayScan VTi (Ceilomics/ThermoFisher),CellularCompartment BioApplication Assay validation/QC 352 top scorers,were rescreened with serial dilutions Correction factors — Normalizationz-score Additional comments Post-HTS analysis Hit criteria Visualinspection Hit rate 68 (0.4%) Additional assay(s) — Confirmation of hitpurity and structure Confirmation of purity and structure was performedfor followed-up hit compounds. (See suppl. methods) Additional comments

TABLE 3 Crystal data and refinement details for the X-ray structuredeterminations of the compound FLI-06. Compound FLI-06 formulaC₂₅H₃₀N₂O₅ fw (g · mol⁻¹) 438.51 T/° C. −140(2) crystal systemmonoclinic space group P2₁/c a/Å 18.0463(9) b/Å 10.4521(5) c/Å12.3077(4) α/° 90.00 β/° 101.616(3) γ/° 90.00 V/Å³  2273.95(17) Z 4 ρ (g· cm⁻³) 1.281 μ (mm⁻¹) .89 measured data 12592 data with I > 2σ(I) 4320unique data (R_(int)) 5142/0.0359 wR₂ (all data, on 0.1261 F²)^(a)) R₁(I > 2σ(I))^(a)) 0.0516 S^(b)) 1.147 Res. dens./e · Å⁻³ 0.255/-0.241absorpt method NONE CCDC No. 911241 ^(a))Definition of the R indices: R₁= (Σ||F_(o)| − |F_(c)||)/Σ|F_(o)|; wR₂ = {Σ[w(F_(o) ² − F_(c)²)²]/Σ[w(F_(o) ²)²]}^(1/2) with w⁻¹ = σ²(F_(o) ²) + (aP)² + bP; P =[2F_(c) ² + Max[Fo²]/3; ^(b))s = {Σ[w(Fo² − F_(c) ²)²]/(N_(o) −N_(p))}^(1/2).

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1. Compound according to the general formula I as an inhibitor of thenotch signalling pathway for use as a medicament for the treatment of asecretion-dependent disease

whereby R1 is one of:

whereby X is H or a halogen, preferably F, Cl, Br or I, and Y is COOCH₃or CF3, or COOH; R2 is a straight chain or branched alkyl group ofC₁-C₈, or a carbon ring structure of C₅-C₈, preferably one of

R3 is H or a straight chain or branched alkyl group of C₁-C₆; R4 is H ora straight chain or branched alkyl group of C₁-C₆, preferably a branchedalkyl group selected from

R5 is H or a straight chain or branched alkyl group of C₁-C₆; whereby R4and R5 may be closed with a C₃ group to form a C₆ ring structure withthe group

whereby R6 and/or R7 is H or a straight chain or branched alkyl group ofC₁-C₆; whereby “R” of the substituents R1, R2, R4, R5 represents thebackbone of general formula I.
 2. Compound according to claim 1 of thegeneral formula II

whereby R1-R3 are as defined by claim 1, and R6 and/or R7 is H or astraight chain or branched alkyl group of C₁-C₆.
 3. Compound accordingto any one of the preceding claims of general formula III

whereby R1: H, COOCH₃, COOH, CN, NO₂, CF₃, or NH₂ and R2: a straightchain or branched alkyl group of C₁-C₈, preferably C₂-C₆, or a carbonring structure, preferably C₅, C₆, C₇ or C₈.
 4. Compound according toany one of the preceding claims with a structure according to any one offormulae I, II or III, whereby R2 to R7 are as defined by claims 1, 2and/or 3 and R1 is


5. Compound according to any one of claims 1 to 4, selected from thegroup comprising of FLI-06, FLI-24, FLI-25, FLI-26, FLI-27 and/or FLI-28as an inhibitor of the notch signalling pathway for use as a medicamentfor the treatment of a secretion-dependent disease


6. Compound according to any one of the preceding claims, whereinsecretion-dependent diseases are selected from those diseases for whichtheir aetiology, pathogenesis, morphologic features and/or clinicalmanifestations are characterised by a dependence on cellular secretionand/or a secretory pathway, and/or by levels of secretion of secretedcompounds, preferably proteins, above normal levels in comparison to ahealthy subject, whereby a therapeutic effect in the treatment of saiddiseases is induced by the inhibition of cellular secretion.
 7. Compoundaccording to the preceding claim, wherein the secretion or secretorypathway is related to, mediated and/or characterised by cytokinesecretion, TNFα secretion, IL-6 secretion, IL-8 secretion, IL-10secretion, Wnt secretion, microRNA secretion, CCL2-secretion, ERtransport and/or the Golgi apparatus.
 8. Compound according to any oneof the preceding claims, wherein the disease is cancer.
 9. Compoundaccording to any one of the preceding claims, wherein the disease issenescence-related ageing.
 10. Compound according to any one of thepreceding claims, wherein the compound does not directly inhibitγ-secretase
 11. Compound according to any one of the preceding claimsfor the treatment of cancer, wherein the cancer is chronic lymphocyticleukemia (CLL), esophageal cancer, glioma, colon cancer or breastcancer.
 12. Compound according to any one of the preceding claims forthe treatment of cancer, whereby the cancer is characterised bymisregulation of the notch signalling pathway.
 13. Compound according toany one of the preceding claims for the treatment of cancer, whereby thecancer is haematological cancer, colorectal cancer, cervical cancer,pancreatic cancer, breast cancer or lung cancer.
 14. Compound accordingto the preceding claim, whereby the haematological cancer is a lymphomaor leukemia.
 15. Compound according to the preceding claim, whereby thelymphoma is a T-cell lymphoma, B-cell lymphoma or Hodgkin lymphoma. 16.Compound according to any one of the preceding claims, whereinsenescence-related ageing is characterised by the presence ofsenescence-associated secretory phenotype (SASP).
 17. Compound accordingto the preceding claim, wherein the SASP is an inflammatory disease. 18.Compound according to the preceding claim, wherein the SASP inflammatorydisease is induced or characterised by secretion of proinflammatorycytokines, in particular IL-6 and IL-8.
 19. Pharmaceutical compositioncomprising one or more of the compounds according to any one of thepreceding claims and a pharmaceutically acceptable carrier substance.20. Use of the compound according to any one of the preceding claims asa lead structure for derivatization in the development of furtherbiologically active compounds.
 21. In vitro use of the compoundaccording to any one of claims 1 to 11 as an inhibitor of the notchsignalling pathway, as an inhibitor of secretion or a secretion pathwayand/or as an inhibitor of membrane traffic.
 22. Method for the treatmentof a secretion-dependent disease, preferably cancer orsenescence-related ageing, more preferably haematological cancer,colorectal cancer, cervical cancer, pancreatic cancer, breast cancer orlung cancer, chronic lymphocytic leukemia (CLL), esophageal cancer,glioma or colon cancer, more preferably lymphoma or leukemia, such asT-cell lymphoma, B-cell lymphoma, Hodgkin lymphoma or inflammatorydisease, comprising administration of an effective amount of apharmaceutical composition comprising one ore more of the compoundsaccording to claims 1 to 5 to a subject in need of treatment.